Influenza virus vaccines and uses thereof

ABSTRACT

Provided herein are influenza hemagglutinin stem domain polypeptides, compositions comprising the same, vaccines comprising the same and methods of their use.

This application is a divisional of U.S. application Ser. No.12/750,393, filed Mar. 30, 2010, issued as U.S. Pat. No. 9,051,359,which claims priority benefit of U.S. Provisional Application No.61/164,896, filed Mar. 30, 2009 and U.S. Provisional Application No.61/299,084, filed Jan. 28, 2010, each of which is incorporated byreference in its entirety herein.

This invention was made with United States Government support underaward number RC1 AI086061 from the National Institutes of Health (NIH)National Institute of Allergy and Infectious Diseases, award number U54AI057158 from the NIH, award number HHSN266200700010C from the UnitedStates Department of Health and Human Services, and award number U01AI070469 from the NIH. The United States Government has certain rightsin this invention.

1. INTRODUCTION

Provided herein are influenza hemagglutinin stem domain polypeptides,compositions comprising the same, vaccines comprising the same andmethods of their use.

2. BACKGROUND

Influenza viruses are enveloped RNA viruses that belong to the family ofOrthomyxoviridae (Palese and Shaw (2007) Orthomyxoviridae: The Virusesand Their Replication, 5th ed. Fields' Virology, edited by B. N. Fields,D. M. Knipe and P. M. Howley. Wolters Kluwer Health/Lippincott Williams& Wilkins, Philadelphia, USA, p 1647-1689). The natural host ofinfluenza viruses are avians, but influenza viruses (including those ofavian origin) also can infect and cause illness in humans and otheranimal hosts (canines, pigs, horses, sea mammals, and mustelids). Forexample, the H5N1 avian influenza virus circulating in Asia has beenfound in pigs in China and Indonesia and has also expanded its hostrange to include cats, leopards, and tigers, which generally have notbeen considered susceptible to influenza A (CIDRAP—Avian Influenza:Agricultural and Wildlife Considerations). The occurrence of influenzavirus infections in animals could potentially give rise to humanpandemic influenza strains.

Influenza A and B viruses are major human pathogens, causing arespiratory disease that ranges in severity from sub-clinical infectionto primary viral pneumonia which can result in death. The clinicaleffects of infection vary with the virulence of the influenza strain andthe exposure, history, age, and immune status of the host. Thecumulative morbidity and mortality caused by seasonal influenza issubstantial due to the relatively high attack rate. In a normal season,influenza can cause between 3-5 million cases of severe illness and upto 500,000 deaths worldwide (World Health Organization (2003) Influenza:Overview). In the United States, influenza viruses infect an estimated10-15% of the population (Glezen and Couch R B (1978) Interpandemicinfluenza in the Houston area, 1974-76. N Engl J Med 298: 587-592; Foxet al. (1982) Influenza virus infections in Seattle families, 1975-1979.II. Pattern of infection in invaded households and relation of age andprior antibody to occurrence of infection and related illness. Am JEpidemiol 116: 228-242) and are associated with approximately 30,000deaths each year (Thompson W W et al. (2003) Mortality Associated withInfluenza and Respiratory Syncytial Virus in the United States. JAMA289: 179-186; Belshe (2007) Translational research on vaccines:influenza as an example. Clin Pharmacol Ther 82: 745-749).

In addition to annual epidemics, influenza viruses are the cause ofinfrequent pandemics. For example, influenza A viruses can causepandemics such as those that occurred in 1918, 1957, 1968, and 2009. Dueto the lack of pre-formed immunity against the major viral antigen,hemagglutinin (HA), pandemic influenza can affect greater than 50% ofthe population in a single year and often causes more severe diseasethan epidemic influenza. A stark example is the pandemic of 1918, inwhich an estimated 50-100 million people were killed (Johnson andMueller (2002) Updating the Accounts: Global Mortality of the 1918-1920“Spanish” Influenza Pandemic Bulletin of the History of Medicine 76:105-115). Since the emergence of the highly pathogenic avian H5N1influenza virus in the late 1990s (Claas et al. (1998) Human influenza AH5N1 virus related to a highly pathogenic avian influenza virus. Lancet351: 472-7), there have been concerns that it may be the next pandemicvirus.

An effective way to protect against influenza virus infection is throughvaccination; however, current vaccination approaches rely on achieving agood match between circulating strains and the isolates included in thevaccine. Such a match is often difficult to attain due to a combinationof factors. First, influenza viruses are constantly undergoing change:every 3-5 years the predominant strain of influenza A virus is replacedby a variant that has undergone sufficient antigenic drift to evadeexisting antibody responses. Isolates to be included in vaccinepreparations must therefore be selected each year based on the intensivesurveillance efforts of the World Health Organization (WHO)collaborating centers. Second, to allow sufficient time for vaccinemanufacture and distribution, strains must be selected approximately sixmonths prior to the initiation of the influenza season. Often, thepredictions of the vaccine strain selection committee are inaccurate,resulting in a substantial drop in the efficacy of vaccination.

The possibility of a novel subtype of influenza A virus entering thehuman population also presents a significant challenge to currentvaccination strategies. Since it is impossible to predict what subtypeand strain of influenza virus will cause the next pandemic, current,strain-specific approaches cannot be used to prepare a pandemicinfluenza vaccine.

3. SUMMARY

In one aspect, provided herein are influenza hemagglutinin stem domainpolypeptides. In certain embodiments, the influenza hemagglutinin stemdomain polypeptides lack globular head domains as described herein.

While not intending to be bound by any particular theory of operation,it is believed that the globular head domain of an influenzahemagglutinin comprises one or more highly immunogenic regions. Thesehighly immunogenic regions might generate a host immune response.However, the highly immunogenic regions might also vary from strain tostrain of influenza virus. Embodiments presented herein are based on, inpart, the discovery that residues in influenza hemagglutinin stemdomains are relatively conserved and immunogenic, and that antibodiesbinding to this region may be neutralizing. An influenza hemagglutininstem domain polypeptide, lacking all or substantially all of aninfluenza hemagglutinin globular head domain, may be used to generate animmune response to one or more conserved epitopes of the stem domainpolypeptide. Removal of the highly immunogenic regions of the globularhead domain might expose one or more epitopes of the stem domainpolypeptide to a host immune system. In addition, in certainembodiments, elimination of the glycosylation of the influenzahemagglutinin stem domain through alteration of glycosylation sitespresent therein may render the conserved regions of the stem domain moreaccessible to the host immune response.

If the one or more epitopes of the stem domain polypeptide are lessimmunogenic than the highly immunogenic regions of a globular headdomain, the absence of a globular head domain in the stem domainpolypeptide might allow an immune response against the one or moreepitopes of the stem domain polypeptide to develop. Advantageously,since the amino acid sequences of influenza hemagglutinin stem domainpolypeptides might be conserved or highly conserved across viralsubtypes, an immune response against an influenza hemagglutinin stemdomain polypeptide provided herein might cross react with one or moreviral subtypes other than the subtype corresponding to the stem domainpolypeptide. Accordingly, the influenza hemagglutinin stem domainpolypeptides provided herein may be useful for immunogenic compositions(e.g. vaccines) capable of generating immune responses against aplurality of influenza virus strains.

Without being bound by any theory, influenza hemagglutinin stem domainpolypeptides described herein are based, in part, on the inventors'discovery of polypeptides that lack the globular head domain ofinfluenza hemagglutinin and maintain the stability of the pre-fusionconformation of influenza hemagglutinin. In one aspect, without beingbound by theory, the inventors have discovered that the maintenance ofcysteine residues identified as A and A in influenza hemagglutininpolypeptides in FIG. 1 contributes the stability of the stalk region ofinfluenza hemagglutinin. In another aspect, without being bound bytheory, the inventors have discovered that influenza hemagglutinin stemdomain polypeptides that maintain the pre-fusion conformation ofinfluenza hemagglutinin polypeptides are more effective at inducing aprotective effect in subjects. In certain aspects, the stability of thepre-fusion conformation can be conferred by introducing amino acidsubstitutions at certain residues, such as HA1 H17Y (H3 numbering).

3.1 Terminology

The terms “about” or “approximate,” when used in reference to an aminoacid position refer to the particular amino acid position in a sequenceor any amino acid that is within five, four, three, two or one residuesof that amino acid position, either in an N-terminal direction or aC-terminal direction.

As used herein, the term “about” or “approximately” when used inconjunction with a number refers to any number within 1, 5 or 10% of thereferenced number.

The term “amino acid sequence identity” refers to the degree of identityor similarity between a pair of aligned amino acid sequences, usuallyexpressed as a percentage. Percent identity is the percentage of aminoacid residues in a candidate sequence that are identical (i.e., theamino acid residues at a given position in the alignment are the sameresidue) or similar (i.e., the amino acid substitution at a givenposition in the alignment is a conservative substitution, as discussedbelow), to the corresponding amino acid residue in the peptide afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence homology. Sequence homology, includingpercentages of sequence identity and similarity, are determined usingsequence alignment techniques well-known in the art, preferably computeralgorithms designed for this purpose, using the default parameters ofsaid computer algorithms or the software packages containing them.Non-limiting examples of computer algorithms and software packagesincorporating such algorithms include the following. The BLAST family ofprograms exemplify a particular, non-limiting example of a mathematicalalgorithm utilized for the comparison of two sequences (e.g., Karlin &Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268 (modified as inKarlin & Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877),Altschul et al., 1990, J. Mol. Biol. 215:403-410, (describing NBLASTand) (BLAST), Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402(describing Gapped BLAST, and PSI-Blast). Another particular example isthe algorithm of Myers and Miller (1988 CABIOS 4:11-17) which isincorporated into the ALIGN program (version 2.0) and is available aspart of the GCG sequence alignment software package. Also particular isthe FASTA program (Pearson W. R. and Lipman D. J., Proc. Nat. Acad. Sci.USA, 85:2444-2448, 1988), available as part of the Wisconsin SequenceAnalysis Package. Additional examples include BESTFIT, which uses the“local homology” algorithm of Smith and Waterman (Advances in AppliedMathematics, 2:482-489, 1981) to find best single region of similaritybetween two sequences, and which is preferable where the two sequencesbeing compared are dissimilar in length; and GAP, which aligns twosequences by finding a “maximum similarity” according to the algorithmof Neddleman and Wunsch (J. Mol. Biol. 48:443-354, 1970), and ispreferable where the two sequences are approximately the same length andan alignment is expected over the entire length.

“Conservative substitution” refers to replacement of an amino acid ofone class is with another amino acid of the same class. In particularembodiments, a conservative substitution does not alter the structure orfunction, or both, of a polypeptide. Classes of amino acids for thepurposes of conservative substitution include hydrophobic (Met, Ala,Val, Leu, Ile), neutral hydrophilic (Cys, Ser, Thr), acidic (Asp, Glu),basic (Asn, Gln, His, Lys, Arg), conformation disrupters (Gly, Pro) andaromatic (Trp, Tyr, Phe).

As used herein, the terms “disease” and “disorder” are usedinterchangeably to refer to a condition in a subject. In someembodiments, the condition is a viral infection. In specificembodiments, a term “disease” refers to the pathological state resultingfrom the presence of the virus in a cell or a subject, or by theinvasion of a cell or subject by the virus. In certain embodiments, thecondition is a disease in a subject, the severity of which is decreasedby inducing an immune response in the subject through the administrationof an immunogenic composition.

As used herein, the term “effective amount” in the context ofadministering a therapy to a subject refers to the amount of a therapywhich has a prophylactic and/or therapeutic effect(s). In certainembodiments, an “effective amount” in the context of administration of atherapy to a subject refers to the amount of a therapy which issufficient to achieve one, two, three, four, or more of the followingeffects: (i) reduce or ameliorate the severity of an influenza virusinfection, disease or symptom associated therewith; ii) reduce theduration of an influenza virus infection, disease or symptom associatedtherewith; (iii) prevent the progression of an influenza virusinfection, disease or symptom associated therewith; (iv) causeregression of an influenza virus infection, disease or symptomassociated therewith; (v) prevent the development or onset of aninfluenza virus infection, disease or symptom associated therewith; (vi)prevent the recurrence of an influenza virus infection, disease orsymptom associated therewith; (vii) reduce or prevent the spread of aninfluenza virus from one cell to another cell, one tissue to anothertissue, or one organ to another organ; (ix) prevent or reduce the spreadof an influenza virus from one subject to another subject; (x) reduceorgan failure associated with an influenza virus infection; (xi) reducehospitalization of a subject; (xii) reduce hospitalization length;(xiii) increase the survival of a subject with an influenza virusinfection or disease associated therewith; (xiv) eliminate an influenzavirus infection or disease associated therewith; (xv) inhibit or reduceinfluenza virus replication; (xvi) inhibit or reduce the entry of aninfluenza virus into a host cell(s); (xviii) inhibit or reducereplication of the influenza virus genome; (xix) inhibit or reducesynthesis of influenza virus proteins; (xx) inhibit or reduce assemblyof influenza virus particles; (xxi) inhibit or reduce release ofinfluenza virus particles from a host cell(s); (xxii) reduce influenzavirus titer; and/or (xxiii) enhance or improve the prophylactic ortherapeutic effect(s) of another therapy.

In certain embodiments, the effective amount does not result in completeprotection from an influenza virus disease, but results in a lower titeror reduced number of influenza viruses compared to an untreated subject.In certain embodiments, the effective amount results in a 0.5 fold, 1fold, 2 fold, 4 fold, 6 fold, 8 fold, 10 fold, 15 fold, 20 fold, 25fold, 50 fold, 75 fold, 100 fold, 125 fold, 150 fold, 175 fold, 200fold, 300 fold, 400 fold, 500 fold, 750 fold, or 1,000 fold or greaterreduction in titer of influenza virus relative to an untreated subject.In some embodiments, the effective amount results in a reduction intiter of influenza virus relative to an untreated subject ofapproximately 1 log or more, approximately 2 logs or more, approximately3 logs or more, approximately 4 logs or more, approximately 5 logs ormore, approximately 6 logs or more, approximately 7 logs or more,approximately 8 logs or more, approximately 9 logs or more,approximately 10 logs or more, 1 to 3 logs, 1 to 5 logs, 1 to 8 logs, 1to 9 logs, 2 to 10 logs, 2 to 5 logs, 2 to 7 logs, 2 logs to 8 logs, 2to 9 logs, 2 to 10 logs 3 to 5 logs, 3 to 7 logs, 3 to 8 logs, 3 to 9logs, 4 to 6 logs, 4 to 8 logs, 4 to 9 logs, 5 to 6 logs, 5 to 7 logs, 5to 8 logs, 5 to 9 logs, 6 to 7 logs, 6 to 8 logs, 6 to 9 logs, 7 to 8logs, 7 to 9 logs, or 8 to 9 logs. Benefits of a reduction in the titer,number or total burden of influenza virus include, but are not limitedto, less severe symptoms of the infection, fewer symptoms of theinfection and a reduction in the length of the disease associated withthe infection.

“Hemagglutinin” and “HA” refer to any hemagglutinin known to those ofskill in the art. In certain embodiments, the hemagglutinin is influenzahemagglutinin, such as an influenza A hemagglutinin, an influenza Bhemagglutinin or an influenza C hemagglutinin. A typical hemagglutinincomprises domains known to those of skill in the art including a signalpeptide (optional herein), a stem domain, a globular head domain, aluminal domain (optional herein), a transmembrane domain (optionalherein) and a cytoplasmic domain (optional herein). In certainembodiments, a hemagglutinin consists of a single polypeptide chain,such as HA0. In certain embodiments, a hemagglutinin consists of morethan one polypeptide chain in quaternary association, e.g. HA1 and HA2.Those of skill in the art will recognize that an immature HA0 might becleaved to release a signal peptide (approximately 20 amino acids)yielding a mature hemagglutinin HA0. A hemagglutinin HA0 might becleaved at another site to yield HA1 polypeptide (approximately 320amino acids, including the globular head domain and a portion of thestem domain) and HA2 polypeptide (approximately 220 amino acids,including the remainder of the stem domain, a luminal domain, atransmembrane domain and a cytoplasmic domain). In certain embodiments,a hemagglutinin comprises a signal peptide, a transmembrane domain and acytoplasmic domain. In certain embodiments, a hemagglutinin lacks asignal peptide, i.e. the hemagglutinin is a mature hemagglutinin. Incertain embodiments, a hemagglutinin lacks a transmembrane domain orcytoplasmic domain, or both. As used herein, the terms “hemagglutinin”and “HA” encompass hemagglutinin polypeptides that are modified bypost-translational processing such as signal peptide cleavage, disulfidebond formation, glycosylation (e.g., N-linked glycosylation), proteasecleavage and lipid modification (e.g. S-palmitoylation).

“HA1 N-terminal stem segment” refers to a polypeptide segment thatcorresponds to the amino-terminal portion of the stem domain of aninfluenza hemagglutinin HA1 polypeptide. In certain embodiments, an HA1N-terminal stem segment consists of amino acid residues correspondingapproximately to amino acids A_(N-term) through A_(p) of an HA1 domain.A_(N-term) is the N-terminal amino acid of HA1 as recognized by those ofskill in the art. A_(p) is the cysteine residue in the HA1 N-terminalstem segment that forms or is capable of forming a disulfide bond with acysteine residue in an HA1 C-terminal stem segment. Residue A_(p) isidentified in influenza A hemagglutinin polypeptides in FIG. 1.Exemplary HA1 N-terminal stem segments are described herein. In certainembodiments, an HA1 N-terminal stem segment consists of amino acidresidues corresponding approximately to amino acids 1-52 of HA1 from anH3 hemagglutinin. Note that, in this numbering system, 1 refers to theN-terminal amino acid of the mature HA0 protein, from which the signalpeptide has been removed.

“HA1 C-terminal stem segment” refers to a polypeptide segment thatcorresponds to the carboxy-terminal portion of the stem domain of aninfluenza hemagglutinin HA1 polypeptide. In certain embodiments, an HA1C-terminal stem segment consists of amino acid residues correspondingapproximately to amino acids A_(q) through A_(C-term) of an HA1 domain.A_(q) is the cysteine residue in the HA1 C-terminal stem segment thatforms or is capable of forming a disulfide bond with a cysteine residuein an HA1 N-terminal stem segment. A_(C-term) is the C-terminal aminoacid of the HA1 domain as recognized by those of skill in the art.Residue A_(q) is identified in influenza A hemagglutinin polypeptides inFIG. 1. Exemplary HA1 C-terminal stem segments are described herein. Incertain embodiments, an HA1 C-terminal stem segment consists of aminoacid residues corresponding approximately to amino acids 277-346 of HA1from an H3 hemagglutinin. Note that, in this numbering system, 1 refersto the N-terminal amino acid of the mature HA0 protein, from which thesignal peptide has been removed.

“HA2” refers to a polypeptide domain that corresponds to the HA2 domainof an influenza hemagglutinin polypeptide known to those of skill in theart. In certain embodiments, an HA2 consists of a stem domain, a luminaldomain, a transmembrane domain and a cytoplasmic domain (see, e.g.,Scheiffle et al., 2007, EMBO J. 16(18):5501-5508, the contents of whichare incorporated by reference in their entirety). In certainembodiments, an HA2 consists of a stem domain, a luminal domain and atransmembrane domain. In certain embodiments, an HA2 consists of a stemdomain and a luminal domain; in such embodiments, the HA2 might besoluble. In certain embodiments, an HA2 consists of a stem domain; insuch embodiments, the HA2 might be soluble.

As used herein, the term “heterologous” in the context of a polypeptide,nucleic acid or virus refers to a polypeptide, nucleic acid or virus,respectively, that is not normally found in nature or not normallyassociated in nature with a polypeptide, nucleic acid or virus ofinterest. For example, a “heterologous polypeptide” may refer to apolypeptide derived from a different virus, e.g., a different influenzastrain or subtype, or an unrelated virus or different species.

As used herein, the term “in combination,” in the context of theadministration of two or more therapies to a subject, refers to the useof more than one therapy (e.g., more than one prophylactic agent and/ortherapeutic agent). The use of the term “in combination” does notrestrict the order in which therapies are administered to a subject. Forexample, a first therapy (e.g., a first prophylactic or therapeuticagent) can be administered prior to (e.g., 5 minutes, 15 minutes, 30minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks,4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantlywith, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks,5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of asecond therapy to a subject.

As used herein, the term “infection” means the invasion by,multiplication and/or presence of a virus in a cell or a subject. In oneembodiment, an infection is an “active” infection, i.e., one in whichthe virus is replicating in a cell or a subject. Such an infection ischaracterized by the spread of the virus to other cells, tissues, and/ororgans, from the cells, tissues, and/or organs initially infected by thevirus. An infection may also be a latent infection, i.e., one in whichthe virus is not replicating. In certain embodiments, an infectionrefers to the pathological state resulting from the presence of thevirus in a cell or a subject, or by the invasion of a cell or subject bythe virus.

As used herein, the term “influenza virus disease” refers to thepathological state resulting from the presence of an influenza (e.g.,influenza A or B virus) virus in a cell or subject or the invasion of acell or subject by an influenza virus. In specific embodiments, the termrefers to a respiratory illness caused by an influenza virus.

As used herein, the phrases “IFN deficient system” or “IFN-deficientsubstrate” refer to systems, e.g., cells, cell lines and animals, suchas pigs, mice, chickens, turkeys, rabbits, rats, etc., which do notproduce IFN or produce low levels of IFN (i.e., a reduction in IFNexpression of 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%,70-80%, 80-90% or more when compared to IFN-competent systems under thesame conditions), do not respond or respond less efficiently to IFN,and/or are deficient in the activity of one or more antiviral genesinduced by IFN.

As used herein, the numeric term “log” refers to log₁₀.

As used herein, the phrase “multiplicity of infection” or “MOI” is theaverage number of infectious virus particles per infected cell. The MOIis determined by dividing the number of infectious virus particles added(ml added×PFU/ml) by the number of cells added (ml added×cells/ml).

As used herein, the term “nucleic acid” is intended to include DNAmolecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) andanalogs of the DNA or RNA generated using nucleotide analogs. Thenucleic acid can be single-stranded or double-stranded.

“Polypeptide” refers to a polymer of amino acids linked by amide bondsas is known to those of skill in the art. As used herein, the term canrefer to a single polypeptide chain linked by covalent amide bonds. Theterm can also refer to multiple polypeptide chains associated bynon-covalent interactions such as ionic contacts, hydrogen bonds, Vander Waals contacts and hydrophobic contacts. Those of skill in the artwill recognize that the term includes polypeptides that have beenmodified, for example by post-translational processing such as signalpeptide cleavage, disulfide bond formation, glycosylation (e.g.,N-linked glycosylation), protease cleavage and lipid modification (e.g.S-palmitoylation).

As used herein, the terms “prevent,” “preventing” and “prevention” inthe context of the administration of a therapy(ies) to a subject toprevent an influenza virus disease refer to one or more of the followingeffects resulting from the administration of a therapy or a combinationof therapies: (i) the inhibition of the development or onset of aninfluenza virus disease or a symptom thereof; (ii) the inhibition of therecurrence of an influenza virus disease or a symptom associatedtherewith; and (iii) the reduction or inhibition in influenza virusinfection and/or replication.

As used herein, the terms “purified” and “isolated” when used in thecontext of a polypeptide (including antibody) that is obtained from anatural source, e.g., cells, refers to a polypeptide which issubstantially free of contaminating materials from the natural source,e.g., soil particles, minerals, chemicals from the environment, and/orcellular materials from the natural source, such as but not limited tocell debris, cell wall materials, membranes, organelles, the bulk of thenucleic acids, carbohydrates, proteins, and/or lipids present in cells.Thus, a polypeptide that is isolated includes preparations of apolypeptide having less than about 30%, 20%, 10%, 5%, 2%, or 1% (by dryweight) of cellular materials and/or contaminating materials. As usedherein, the terms “purified” and “isolated” when used in the context ofa polypeptide (including antibody) that is chemically synthesized refersto a polypeptide which is substantially free of chemical precursors orother chemicals which are involved in the syntheses of the polypeptide.In a specific embodiment, an influenza hemagglutinin stem domainpolypeptide is chemically synthesized. In another specific embodiment,an influenza hemagglutinin stem domain polypeptide is isolated.

As used herein, the terms “replication,” “viral replication” and “virusreplication” in the context of a virus refer to one or more, or all, ofthe stages of a viral life cycle which result in the propagation ofvirus. The steps of a viral life cycle include, but are not limited to,virus attachment to the host cell surface, penetration or entry of thehost cell (e.g., through receptor mediated endocytosis or membranefusion), uncoating (the process whereby the viral capsid is removed anddegraded by viral enzymes or host enzymes thus releasing the viralgenomic nucleic acid), genome replication, synthesis of viral messengerRNA (mRNA), viral protein synthesis, and assembly of viralribonucleoprotein complexes for genome replication, assembly of virusparticles, post-translational modification of the viral proteins, andrelease from the host cell by lysis or budding and acquisition of aphospholipid envelope which contains embedded viral glycoproteins. Insome embodiments, the terms “replication,” “viral replication” and“virus replication” refer to the replication of the viral genome. Inother embodiments, the terms “replication,” “viral replication” and“virus replication” refer to the synthesis of viral proteins.

“Stem domain polypeptide” refers to a derivative, e.g. an engineeredderivative, of a hemagglutinin polypeptide that comprises one or morepolypeptide chains that make up a stem domain of hemagglutinin. A stemdomain polypeptide might be a single polypeptide chain, two polypeptidechains or more polypeptide chains. Typically, a stem domain polypeptideis a single polypeptide chain (i.e. corresponding to the stem domain ofa hemagglutinin HA0 polypeptide) or two polypeptide chains (i.e.corresponding to the stem domain of a hemagglutinin HA1 polypeptide inassociation with a hemagglutinin HA2 polypeptide). In certainembodiments, a stem domain polypeptide is derived from an influenzahemagglutinin. Engineered stem domain polypeptides can comprise one ormore linkers as described below.

As used herein, the terms “subject” or “patient” are usedinterchangeably to refer to an animal (e.g., birds, reptiles, andmammals). In a specific embodiment, a subject is a bird. In anotherembodiment, a subject is a mammal including a non-primate (e.g., acamel, donkey, zebra, cow, pig, horse, goat, sheep, cat, dog, rat, andmouse) and a primate (e.g., a monkey, chimpanzee, and a human). Incertain embodiments, a subject is a non-human animal. In someembodiments, a subject is a farm animal or pet. In another embodiment, asubject is a human. In another embodiment, a subject is a human infant.In another embodiment, a subject is a human child. In anotherembodiment, a subject is a human adult. In another embodiment, a subjectis an elderly human. In another embodiment, a subject is a prematurehuman infant.

As used herein, the term “premature human infant” refers to a humaninfant born at less than 37 weeks of gestational age.

As used herein, the term “human infant” refers to a newborn to 1 yearold human.

As used herein, the term “human child” refers to a human that is 1 yearto 18 years old.

As used herein, the term “human adult” refers to a human that is 18years or older.

As used herein, the term “elderly human” refers to a human 65 years orolder.

The terms “tertiary structure” and “quaternary structure” have themeanings understood by those of skill in the art. Tertiary structurerefers to the three-dimensional structure of a single polypeptide chain.Quaternary structure refers to the three dimensional structure of apolypeptide having multiple polypeptide chains.

As used herein, the terms “therapies” and “therapy” can refer to anyprotocol(s), method(s), compound(s), composition(s), formulation(s),and/or agent(s) that can be used in the prevention or treatment of aviral infection or a disease or symptom associated therewith. In certainembodiments, the terms “therapies” and “therapy” refer to biologicaltherapy, supportive therapy, and/or other therapies useful in treatmentor prevention of a viral infection or a disease or symptom associatedtherewith known to one of skill in the art. In some embodiments, theterm “therapy” refers to a nucleic acid encoding an influenza virushemagglutinin stem domain polypeptide, an influenza virus hemagglutininstem domain polypeptide, or a vector or composition comprising saidnucleic acid encoding an influenza virus hemagglutinin stem domainpolypeptide or an influenza hemagglutinin stem domain polypeptide. Insome embodiments, the term “therapy” refers to an antibody thatspecifically binds to an influenza virus hemagglutinin polypeptide or aninfluenza virus hemagglutinin stem domain polypeptide.

As used herein, the terms “treat,” “treatment,” and “treating” refer inthe context of administration of a therapy(ies) to a subject to treatingan influenza virus disease to obtain a beneficial or therapeutic effectof a therapy or a combination of therapies. In specific embodiments,such terms refer to one, two, three, four, five or more of the followingeffects resulting from the administration of a therapy or a combinationof therapies: (i) the reduction or amelioration of the severity of aninfluenza virus infection or a disease or a symptom associatedtherewith; (ii) the reduction in the duration of an influenza virusinfection or a disease or a symptom associated therewith; (iii) theregression of an influenza virus infection or a disease or a symptomassociated therewith; (iv) the reduction of the titer of an influenzavirus; (v) the reduction in organ failure associated with an influenzavirus infection or a disease associated therewith; (vi) the reduction inhospitalization of a subject; (vii) the reduction in hospitalizationlength; (viii) the increase in the survival of a subject; (ix) theelimination of an influenza virus infection or a disease or symptomassociated therewith; (x) the inhibition of the progression of aninfluenza virus infection or a disease or a symptom associatedtherewith; (xi) the prevention of the spread of an influenza virus froma cell, tissue, organ or subject to another cell, tissue, organ orsubject; (xii) the inhibition or reduction in the entry of an influenzavirus into a host cell(s); (xiii) the inhibition or reduction in thereplication of an influenza virus genome; (xiv) the inhibition orreduction in the synthesis of influenza virus proteins; (xv) theinhibition or reduction in the release of influenza virus particles froma host cell(s); and/or (xvi) the enhancement or improvement thetherapeutic effect of another therapy.

As used herein, in some embodiments, the phrase “wild-type” in thecontext of a virus refers to the types of a virus that are prevalent,circulating naturally and producing typical outbreaks of disease. Inother embodiments, the term “wild-type” in the context of a virus refersto a parental virus.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A, FIG. 1B, and FIG. 1C present a sequence alignment by CLUSTALWof representative sequences of 16 subtypes of influenza virus Ahemagglutinin (SEQ ID NOS:1-16, respectively). Due to size limitations,FIG. 1 is divided into three parts: FIG. 1A, FIG. 1B, and FIG. 1C. FIG.1A presents a sequence alignment by CLUSTALW of representative sequencesof 16 subtypes of influenza virus A hemagglutinin (SEQ ID NOS:1-16,respectively). FIG. 1B presents a sequence alignment by CLUSTALW ofrepresentative sequences of 16 subtypes of influenza virus Ahemagglutinin (SEQ ID NOS:1-16, respectively). FIG. 1C presents asequence alignment by CLUSTALW of representative sequences of 16subtypes of influenza virus A hemagglutinin (SEQ ID NOS:1-16,respectively).

FIG. 2 presents a sequence alignment by CLUSTALW of a representativesequence of influenza virus B hemagglutinin (SEQ ID NO:17) aligned withinfluenza A HK68-H3N2 (SEQ ID NO:3) and PR8-H1N1 (SEQ ID NO:1)hemagglutinins.

FIG. 3 provides exemplary nucleotide constructs encoding wild type HAand influenza HA stem domain polypeptides.

FIG. 4 provides the putative structure of an influenza HA stem domainpolypeptide; linker GGGG corresponds to SEQ ID NO:319 and linkerITPNGSIPNDKPFQNVNKITYGA corresponds to SEQ ID NO:165.

FIG. 5A provides protein expression of exemplary influenza HA stemdomain polypeptides. FIG. 5B provides protein expression of exemplaryinfluenza HA stem domain polypeptides.

FIG. 6A and FIG. 8B provide an exemplary construct for expressing aninfluenza HA stem domain polypeptide with nucleotide (SEQ ID NO:169) andamino acid (SEQ ID NO:170) sequences. The glycine linker is underlined.Due to size limitations, FIG. 6 is divided into two parts: FIG. 6A andFIG. 6B. FIG. 6A provides an exemplary construct for expressing aninfluenza HA stem domain polypeptide with nucleotide (SEQ ID NO:169) andamino acid (SEQ ID NO:170) sequences; the glycine linker is underlined.FIG. 6B provides an exemplary construct for expressing an influenza HAstem domain polypeptide with nucleotide (SEQ ID NO:169) and amino acid(SEQ ID NO:170) sequences.

FIG. 7A and FIG. 7B provide an exemplary construct for expressing aninfluenza HA stem domain polypeptide with nucleotide (SEQ ID NO:171) andamino acid (SEQ ID NO:172) sequences. The glycine linker is underlined.Due to size limitations, FIG. 7 is divided into two parts: FIG. 7A andFIG. 7B. FIG. 7A provides an exemplary construct for expressing aninfluenza HA stem domain polypeptide with nucleotide (SEQ ID NO:171) andamino acid (SEQ ID NO:172) sequences; the glycine linker is underlined.FIG. 7B provides an exemplary construct for expressing an influenza HAstem domain polypeptide with nucleotide (SEQ ID NO:171) and amino acid(SEQ ID NO:172) sequences.

FIG. 8A and FIG. 8B provide an exemplary construct for expressing aninfluenza HA stem domain polypeptide with nucleotide (SEQ ID NO:173) andamino acid (SEQ ID NO:174) sequences. The proline-glycine linker isunderlined. Due to size limitations, FIG. 8 is divided into two parts:FIG. 8A and FIG. 8B. FIG. 8A provides an exemplary construct forexpressing an influenza HA stem domain polypeptide with nucleotide (SEQID NO:173) and amino acid (SEQ ID NO:174) sequences; the proline-glycinelinker is underlined. FIG. 8B provides an exemplary construct forexpressing an influenza HA stem domain polypeptide with nucleotide (SEQID NO:173) and amino acid (SEQ ID NO:174) sequences.

FIG. 9A and FIG. 9B provide an exemplary construct for expressing aninfluenza HA stem domain polypeptides with nucleotide (SEQ ID NO:175)and amino acid (SEQ ID NO:176) sequences. The glycine linker, thrombincleavage site, foldon domain and HIS tag are underlined. Due to sizelimitations, FIG. 9 is divided into two parts: FIG. 9A and FIG. 9B. FIG.9A provides an exemplary construct for expressing an influenza HA stemdomain polypeptides with nucleotide (SEQ ID NO:175) and amino acid (SEQID NO:176) sequences; the glycine linker is underlined. FIG. 9B providesan exemplary construct for expressing an influenza HA stem domainpolypeptides with nucleotide (SEQ ID NO:175) and amino acid (SEQ IDNO:176) sequences; the thrombin cleavage site, foldon domain and HIS tagare underlined.

FIG. 10A and FIG. 10B. Schematic of headless HA constructs. FIG. 10A:Schematic of the linear structure of the full length influenza virus HAprotein (top) and a generalized headless HA protein (bottom). Linkerpeptides tested in the context of the PR8 and HK68 HA sequences areshown: KLCGGCNTK (SEQ ID NO:321); KLCGGGGCNTK (SEQ ID NO:322); KLCPGCNTK(SEQ ID NO:323); KLGNTK (SEQ ID NO:324); KLGGNTK (SEQ ID NO:325);KLGGGNTK (SEQ ID NO:326); KLNTK (SEQ ID NO:327); KLTK (SEQ ID NO:328);KK, KLNASNTK (SEQ ID NO:329); KICGGCISE (SEQ ID NO:330); KICGGGGCISE(SEQ ID NO:331); KECPGCISE (SEQ ID NO:332); KIISE (SEQ ID NO:333); KISE(SEQ ID NO:334); KE; and KINASNTK (SEQ ID NO:335). Inserted amino acidsare shown in bold face font, while amino acids present in the native HAsequence are in regular font. FIG. 10B: Schematic of the foldedstructures of the full length and headless HAs of PR8 virus (left panel)and HK68 virus (right panel). In both cases headless HAs carrying the 4Glinker bridge are depicted. The HA1 subunit is colored dark grey and theHA2 subunit is light grey. The location of 4G linker sequences isindicated with an arrow in each panel. The full length HA structureswere downloaded from the Protein Database (PDB): PR8 HA, PDB ID 1rvx andHK68 HA, PDB ID 1mgn. Schematics of headless HAs were generated usingthe full length HA coordinates as a starting point and 4G loops weremanually docked into the headless HA carbon to close the discontinuousalpha carbon amino acid chain. Final images were generated by PyMol(Delano Scientific).

FIG. 11A and FIG. 11B. Expression of headless HA constructs intransiently transfected cells. Headless HA constructs were expressed in293T cells by plasmid transfection in the absence of exogenous trypsin.At 24 hours post-transfection, whole cell lysates were prepared andsubjected to SDS-PAGE followed by Western blotting. HA proteins weredetected using the polyclonal 3951 antiserum (for PR8) or the monoclonal12D1 (for HK68). Molecular weight markers in kDa are shown to the leftof each blot and transfected constructs are identified above theappropriate lane. “Mock” indicates untransfected cells; “Full” indicatesthe full length HA protein; for the headless HA constructs, the aminoacid sequence bridging the N and C terminal strands of HA1 is shown:KLCGGCNTK (SEQ ID NO:321); KLCGGGGCNTK (SEQ ID NO:322); KLCPGCNTK (SEQID NO:323); KLGNTK (SEQ ID NO:324); KLGGNTK (SEQ ID NO:325); KLGGGNTK(SEQ ID NO:326); KLNTK (SEQ ID NO:327); KLTK (SEQ ID NO:328); KK,KLNASNTK (SEQ ID NO:329); KICGGCISE (SEQ ID NO:330); KICGGGGCISE (SEQ IDNO:331); KICPGCISE (SEQ ID NO:336); KIISE (SEQ ID NO:333); KISE (SEQ IDNO:334); KE; and KLNASNTK (SEQ ID NO:337). Letters in bold font indicateinserted amino acids, while letters in regular font represent residuespresent in the wild-type HA. In the region of the cys52 to cys277disulfide bond, the wild-type sequences are as follows. PR8: K50L51C52 .. . C277N278T279K280. HK68: K50I51C52 . . . C277I278S279E280. PR8 basedconstructs are shown in panel FIG. 11A and HK68 based constructs areshown in panel FIG. 11B. In FIG. 11B, the molecular weight of the fulllength HK68 HA0 protein is indicated by an arrowhead.

FIG. 12A and FIG. 12B. Detection of headless HA proteins on the surfaceof transfected cells. Full length and headless HA constructs wereexpressed in 293T cells by plasmid transfection. At 24 hourspost-transfection, cells were trypsinized and HA proteins on the cellsurface were stained using the polyclonal 3951 antiserum (for PR8) orthe monoclonal 12D1 (for HK68) prior to analysis by flow cytometry. FIG.12A: Mock transfected cells stained with 3951 immune sera are comparedto cells transfected with pDZ PR8 HA or cells transfected with pCAGGSPR8 2G, 4G or PG headless HA constructs. FIG. 12: Mock transfected cellsstained with mAb 12D1 are compared to cells transfected with pCAGGS HK68HA or cells transfected with pCAGGS HK68 2G, 4G or PG headless HAconstructs.

FIG. 13A and FIG. 13B. Incorporation of headless HA proteins intovirus-like particles. The HA content of VLPs generated byco-transfection of HA constructs with pGagEGFP was assessed by Westernblotting. FIG. 13A: PR8 based VLPs were probed with the polyclonal 3951antiserum. FIG. 13B: HK68 based proteins were detected with themonoclonal 12D1. Bands are identified to the right of each blot. Notethat VLPs were produced in the presence of exogenous trypsin resultingin the cleavage of HA0 to produce HA1 (not visualized here) and HA2.Ramps above the lanes indicate a ⅓ dilution of the sample: for each VLP,the left lane shows VLPs harvested from the equivalent of three 10 cmdishes of 293T cells, while the right lane shows VLPs harvested from one10 cm dish.

FIG. 14. Vaccination of mice with headless HA constructs providesprotection from death. The average body weight loss in each group ofvaccinated mice following challenge with PR8 virus is shown. Error barsrepresent standard deviation. * indicates the death of a mouse.

FIG. 15A, FIG. 15B, FIG. 15C, FIG. 15D, FIGS. 15E, and 15F depict ELISAdata. Anti-sera from mice vaccinated with the PR8 4G headless HA showsbroad cross-reactivity by ELISA. The vaccine groups from which sera arederived are identified at the top of each column and the ELISA substrateused is indicated to the right of each row. Sera from vaccinated miceare shown in black with filled symbols. Each mouse is represented by aunique symbol which is the same in each panel. A rabbit anti-serumraised against whole PR8 virus is shown in grey with open triangles anda serum sample taken from a naïve mouse is shown in grey with opensquares. Reactivity of mouse sera to FIG. 15A whole PR8 virus, FIG. 15Bpurified recombinant A/New Caledonia/20/1999 HA protein, FIG. 15Cpurified recombinant A/California/04/2009 HA, FIG. 15D purifiedrecombinant A/Singapore/1/1957 HA, FIG. 15E purified recombinant A/VietNam/1203/2004 HA, and FIG. 15F purified recombinant A/Hong Kong/1/1968HA are shown.

FIG. 16A and FIG. 16B present schematic diagrams of representativeheadless molecules. FIG. 16A: Headless HA construct based on the A/HongKong/68 hemagglutinin protein with the linker bridge positioned betweenamino acids 52 and 277 of the HA1 domain. FIG. 16B: Headless HAconstruct based on the A/PR/8/34 hemagglutinin protein, with the linkerbridge positioned between amino acids 46 and 276 of the HA1 domain.

FIG. 17A, FIG. 17B, and FIG. 17C present schematic diagrams of theprimary protein sequences of representative HA molecules. FIG. 17A:presents a schematic diagram of the primary protein sequence of wt HA.FIG. 17B presents a schematic diagram of the primary protein sequencesof a representative headless molecule based on the A/Hong Kong/68hemagglutinin protein with the linker bridge positioned between aminoacids 52 and 277 of the HA1 domain. FIG. 17C presents a schematicdiagram of the primary protein sequences of a representative headlessmolecule based on the A/PR/8/34 hemagglutinin protein, with the linkerbridge positioned between amino acids 46 and 276 of the HA1 domain.

5. DETAILED DESCRIPTION 5.1 Polypeptides

Provided herein are influenza hemagglutinin stem domain polypeptides.While not intending to be bound by any particular theory of operation,it is believed that the influenza hemagglutinin stem domain polypeptidesare useful for presenting one or more relatively conserved antigenicregions to a host immune system in order to generate an immune responsethat is capable of cross-reacting with a plurality of influenza strains.Since the one or more antigenic regions are well conserved acrossinfluenza hemagglutinin subtypes, such an immune response mightcross-react with several subtypes of full-length influenza hemagglutininpolypeptides.

It is believed that full-length influenza hemagglutinin presents severalhighly antigenic segments in its globular head domain. These highlyantigenic segments might be more accessible to a host immune system ormore immunogenic in structure, or both. It is believed that a hostimmune system responds preferentially to these highly immunogenicsegments compared to one or more epitopes in the stem domain of aninfluenza hemagglutinin. Further, since a globular head domain of aninfluenza hemagglutinin might be variable across subtypes and viralstrains, an immune response against one globular head domain subtypemight be limited to the specific highly antigenic segments of thatglobular head domain. Strains with different globular head domains mightnot cross react with the same immune response. As such, theeffectiveness of vaccines presenting hemagglutinin polypeptides might belimited to the specific strains presented in the vaccine. Hence, a givenconventional influenza vaccine is likely only effective against theinfluenza strains predicted to be virulent during a given flu season.

Advantageously, influenza hemagglutinin stem domain polypeptidesprovided herein might be useful to generate an immune response againstmultiple influenza strains. The influenza hemagglutinin stem domainpolypeptides generally do not comprise the highly antigenic, variableglobular head domains of conventional influenza vaccine polypeptides.Thus, they should not generate immune responses limited to the variablesegments of the globular head domains. Instead, they present one or moreepitopes in the relatively conserved stem domain of influenzahemagglutinin. As such, they might be used to generate a host immuneresponse against multiple influenza strains that carry the relativelyconserved epitopes. Accordingly, the influenza hemagglutinin stem domainpolypeptides find use as antigens in the compositions, vaccines andmethods described in detail below. The influenza hemagglutinin stemdomain polypeptides might be useful for generating a host immuneresponse against any one, two, three, four, five, six, seven, eight,nine, ten, eleven, twelve, thirteen, fourteen, fifteen or sixteen knowninfluenza A hemagglutinin subtypes or a later identified influenza Ahemagglutinin subtype. The influenza hemagglutinin stem domainpolypeptides might also be useful for generating a host immune responseagainst any influenza B hemagglutinin subtype now known or lateridentified.

Generally, the influenza hemagglutinin stem domain polypeptides providedherein are polypeptides that comprise or consist essentially of the stemdomain of an influenza hemagglutinin polypeptide. The stem domain of aninfluenza hemagglutinin polypeptide is the stem domain that is generallyrecognized by those of skill in the art.

As is known to those of skill in the art, a full-length influenzahemagglutinin typically comprises an HA1 domain and an HA2 domain. Thestem domain is formed by two segments of the HA1 domain and most or allof the HA2 domain. The two segments of the HA1 domain are separated, inprimary sequence, by a globular head domain.

In certain embodiments, influenza hemagglutinin stem domain polypeptidescomprise little or no globular head domain of an influenza hemagglutininpolypeptide. In certain embodiments, an influenza hemagglutinin stemdomain polypeptides is an influenza hemagglutinin that has had itsglobular head domain deleted by any technique deemed suitable by one ofskill in the art.

In certain embodiments, influenza hemagglutinin stem domain polypeptidesdescribed herein maintain the cysteine residues identified in influenzahemagglutinin polypeptides as A_(p) and A_(q) in FIG. 1. In certainembodiments, influenza hemagglutinin stem domain polypeptides describedherein have greater stability at a pH lower than the hemagglutinin of awild-type influenza viruses (e.g., a pH less than 5.2, less than 5.1,less than 5.0, or less than 4.9, such as 4.8, 4.7, 4.6, 4.5, 4.4, 4.3,4.2, 4.1, 4.0, 3.9, 3.8, etc.). In particular embodiments, influenzahemagglutinin stem domain polypeptides described herein undergoconformational changes from the pre-fusion to the fusion conformation ata pH lower than the hemagglutinin of wild-type influenza viruses. Insome embodiments, influenza hemagglutinin stem domain polypeptidesdescribed herein comprise one or more amino acid substitutions, such asHA1 H17Y (H3 numbering) that increases the stability of the polypeptidesat a low pH (e.g., a pH of between 4.9 to 5.2, 4.5 to 3.5, 3.5 to 2.5,2.5 to 1.5, 1.5 to 0.5). The stability of influenza hemagglutinin stemdomain polypeptides can be assessed using techniques known in the art,such as sensitivity of the hemagglutininmolecules to trypsin digestion,as described in, e.g., Thoennes et al., 2008, Virology 370: 403-414.

The influenza hemagglutinin stem domain polypeptides can be preparedaccording to any technique deemed suitable to one of skill, includingtechniques described below. In certain embodiments, the stem domainpolypeptides are isolated.

The typical primary structure of an influenza hemagglutinin stem domainpolypeptide provided herein comprises, in the following order, an HA1N-terminal stem segment, a linker, an HA1 C-terminal stem segment and anHA2. The primary sequence might be formed by a single polypeptide, or itmight be formed by multiple polypeptides. Typically, a singlepolypeptide is expressed by any technique deemed suitable by one ofskill in the art. In single polypeptide embodiments, the HA1 segmentsand the HA2 are in tertiary association. As is known to those of skillin the art, a single HA polypeptide might be cleaved, for example by aprotease, under appropriate expression conditions to yield twopolypeptides in quaternary association. The cleavage is typicallybetween the HA1 C-terminal stem segment and the HA2. In certainembodiments, provided herein are multiple polypeptide, for example twopolypeptide, influenza hemagglutinin stem domains. In multiplepolypeptide embodiments, the HA1 segments and HA2 are in quaternaryassociation.

In certain embodiments, an influenza hemagglutinin stem domainpolypeptide provided herein is monomeric. In certain embodiments, aninfluenza hemagglutinin stem domain polypeptide provided herein ismultimeric. In certain embodiments, an influenza hemagglutinin stemdomain polypeptide provided herein is trimeric. Those of skill in theart will recognize that native influenza hemagglutinin polypeptides arecapable of trimerization in vivo and that certain influenzahemagglutinin stem domain polypeptides provided herein are capable oftrimerization. In particular embodiments described below, influenzahemagglutinin stem domain polypeptides provided herein comprisetrimerization domains to facilitate trimerization.

In certain embodiments, an influenza hemagglutinin stem domainpolypeptide comprises a signal peptide. Typically, the signal peptide iscleaved during or after polypeptide expression and translation to yielda mature influenza hemagglutinin stem domain polypeptide. The signalpeptide might be advantageous for expression of the influenzahemagglutinin stem domain polypeptides. In certain embodiments, alsoprovided herein are mature influenza hemagglutinin stem domainpolypeptides that lack a signal peptide.

Influenza hemagglutinin HA2 typically comprises a stem domain,transmembrane domain and a cytoplasmic domain. In certain embodiments,provided herein are influenza hemagglutinin stem domain polypeptidesthat comprise an HA2 stem domain, an HA2 luminal domain, an HA2transmembrane domain and an HA2 cytoplasmic domain. Such influenzahemagglutinin stem domain polypeptides might be expressed asmembrane-bound antigens. In certain embodiments, provided herein areinfluenza hemagglutinin stem domain polypeptides that comprise an HA2stem domain, an HA2 luminal domain, and an HA2 transmembrane domain butlack some or all of the typical cytoplasmic domain. Such influenzahemagglutinin stem domain polypeptides might be expressed asmembrane-bound antigens. In certain embodiments, provided herein areinfluenza hemagglutinin stem domain polypeptides that comprise an HA2stem domain and an HA2 luminal domain but lack both an HA2 transmembranedomain and an HA2 cytoplasmic domain. Such influenza hemagglutinin stemdomain polypeptides might advantageously be expressed as solublepolypeptides. In certain embodiments, provided herein are influenzahemagglutinin stem domain polypeptides that comprise an HA2 stem domainbut lack an HA2 luminal domain, an HA2 transmembrane domain and an HA2cytoplasmic domain. Such influenza hemagglutinin stem domainpolypeptides might advantageously be expressed as soluble polypeptides.In certain embodiments, the influenza hemagglutinin stem domainpolypeptides comprise an HA2 stem domain having at least 70%, 75%, 80%,85%, 90%, 95%, 96% or 98% amino acid sequence identity to an influenzaHA2 stem domain known to those of skill in the art. Exemplary known HA2stem domains from known influenza A and influenza B hemagglutinins areprovided in the tables below.

Also provided herein are influenza hemagglutinin stem domainpolypeptides comprising deleted forms of HA2 stem domains wherein up to10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residues are deleted fromeither or both termini of the HA2 stem domain. Further provided hereinare influenza hemagglutinin stem domain polypeptides comprising alteredforms of HA2 stem domains wherein up to 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1amino acid residues are conservatively substituted with other aminoacids. Further provided are influenza hemagglutinin stem domainpolypeptides comprising deleted and altered HA2 stem domains.

The HA1 N-terminal stem segment might be any HA1 N-terminal stem segmentrecognized by one of skill in the art based on the definition providedherein. Typically, an HA1 N-terminal stem segment corresponds to apolypeptide consisting of the N-terminal amino acid of a mature HA1(i.e. an HA1 lacking a signal peptide) through the cysteine residuelocated in sequence at approximately the 52^(nd) residue of the HA1.This cysteine residue, termed A_(p) herein, is generally capable offorming a disulfide bridge with a cysteine residue in the C-terminalstem segment of HA1. Sequences of 16 representative influenza Ahemagglutinins are presented in FIG. 1, and residue A_(p) is identifiedin each.

In certain embodiments, the HA1 N-terminal stem segment does not endexactly at A_(p) (e.g., Cys₅₂ of an HA1 subunit from an H3hemagglutinin), but at a residue in sequence and structure vicinity toA_(p). For example, in certain embodiments, the HA1 N-terminal stemsegment ends at A_(p−1), A_(p−2), A_(p−3), or A_(p−4). In otherembodiments, the HA1 N-terminal stem segment ends at A_(p+1), A_(p+2),A_(p+3), A_(p+4) or A_(p+5). The end of an HA1 N-terminal stem segmentshould be selected in conjunction with the end of the HA1 C-terminalstem segment and the linker so that the resulting linked HA1 stem domainis capable of forming a three-dimensional structure similar, asdescribed below, to an influenza hemagglutinin stem domain.

In certain embodiments, the influenza hemagglutinin stem domainpolypeptides comprise an HA1 N-terminal stem segment having at least70%, 75%, 80%, 85%, 90%, 95%, 96% or 98% amino acid sequence identity toan influenza HA1 N-terminal stem segment known to those of skill in theart. Exemplary known HA1 N-terminal stem segments are provided in thetables below.

Also provided herein are influenza hemagglutinin stem domainpolypeptides comprising deleted forms of HA1 N-terminal stem segmentswherein up to 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residues aredeleted from either or both termini of the HA1 N-terminal stem segment.In certain embodiments, provided herein are influenza hemagglutinin stemdomain polypeptides that comprise expanded forms of HA1 N-terminal stemsegments wherein 1, 2 or 3 residues are added to the C-terminus of theHA1 N-terminal stem segments; these added residues might be derived fromthe amino acid sequence of a globular head domain adjacent to an HA1N-terminal stem segment. Further provided herein are influenzahemagglutinin stem domain polypeptides comprising altered forms of HA1N-terminal stem segments wherein up to 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1amino acid residues are conservatively substituted with other aminoacids. Further provided are influenza hemagglutinin stem domainpolypeptides comprising deleted and altered HA1 N-terminal stemsegments.

The HA1 C-terminal stem segment might be any HA1 C-terminal stem segmentrecognized by one of skill in the art based on the definition providedherein. Typically, an HA1 C-terminal stem segment corresponds to apolypeptide consisting of the cysteine residue located in sequence atapproximately the 277^(th) residue of an HA1 (using H3 numbering)through the C-terminal amino acid of the HA1. This cysteine residue,termed A_(q) herein, is generally capable of forming a disulfide bridgewith cysteine residue A_(p) in the N-terminal stem segment of HA1.Sequences of 16 representative influenza A hemagglutinins are presentedin FIG. 1, and residue A_(q) is identified in each.

In certain embodiments, the HA1 C-terminal stem segment does not startat A_(q) (e.g., Cys₂₇₇ of an HA1 subunit from an H3 hemagglutinin), butat a residue in sequence and structure vicinity to A_(q). For example,in certain embodiments, the HA1 C-terminal stem segment starts atA_(q−1), A_(q−2), A_(q−3), or A_(q−4). In other embodiments, the HA1C-terminal stem segment starts at A_(q+1), A_(q+2), A_(q+3), A_(q+4) orA_(q+5). The end of an HA1 N-terminal stem segment should be selected inconjunction with the start of the HA1 C-terminal stem segment and thelinker so that the resulting HA1 stem domain is capable of forming athree-dimensional structure similar, as described below, to an influenzahemagglutinin.

In certain embodiments, the influenza hemagglutinin stem domainpolypeptides comprise an HA1 C-terminal stem segment having at least70%, 75%, 80%, 85%, 90%, 95%, 96% or 98% amino acid sequence identity toan influenza HA1 C-terminal stem segment known to those of skill in theart. Exemplary known HA1 C-terminal stem segments are provided in thetables below.

In certain embodiments, the end of the N-terminal stem segment isA_(p−1), and the start of the C-terminal stem segment is A_(q−1). Incertain embodiments, the end of the N-terminal stem segment is A_(p−2),and the start of the C-terminal stem segment is A_(q−2) In certainembodiments, the end of the N-terminal stem segment is A_(q−3), and thestart of the C-terminal stem segment is A_(q−3) In certain embodiments,the end of the N-terminal stem segment is A_(p−4), and the start of theC-terminal stem segment is A_(q−4) In certain embodiments, the end ofthe N-terminal stem segment is A_(p−5), and the start of the C-terminalstem segment is A_(q−5).

In certain embodiments, the end of the N-terminal stem segment isA_(p+1), and the start of the C-terminal stem segment is A_(q+1) Incertain embodiments, the end of the N-terminal stem segment is A_(p+2),and the start of the C-terminal stem segment is A_(q+2) In certainembodiments, the end of the N-terminal stem segment is A_(p+3), and thestart of the C-terminal stem segment is A_(q+3) In certain embodiments,the end of the N-terminal stem segment is A_(p+4), and the start of theC-terminal stem segment is A_(q+4) In certain embodiments, the end ofthe N-terminal stem segment is A_(p+5), and the start of the C-terminalstem segment is A_(q+5).

In certain embodiments, the end of the N-terminal stem segment isA_(p−1), and the start of the C-terminal stem segment is A_(q+1) Incertain embodiments, the end of the N-terminal stem segment is A_(p−2),and the start of the C-terminal stem segment is A_(q+2) In certainembodiments, the end of the N-terminal stem segment is A_(p−3), and thestart of the C-terminal stem segment is A_(q+3) In certain embodiments,the end of the N-terminal stem segment is A_(p−4), and the start of theC-terminal stem segment is A_(q+4) In certain embodiments, the end ofthe N-terminal stem segment is A_(p−5), and the start of the C-terminalstem segment is A_(q+5).

In certain embodiments, the end of the N-terminal stem segment is A_(p)(i.e., the end of the N-terminal stem segment is Cysteine), and thestart of the C-terminal stem segment is A_(q) (i.e., the start of theC-terminal stem segment is Cysteine) In certain embodiments, the end ofthe N-terminal stem segment is A_(p+1), and the start of the C-terminalstem segment is A_(q−1). In certain embodiments, the end of theN-terminal stem segment is A_(p+2), and the start of the C-terminal stemsegment is A_(q−2) In certain embodiments, the end of the N-terminalstem segment is A_(p+3), and the start of the C-terminal stem segment isA_(q−3) In certain embodiments, the end of the N-terminal stem segmentis A_(p+4), and the start of the C-terminal stem segment is A_(q−4) Incertain embodiments, the end of the N-terminal stem segment is A_(p+5),and the start of the C-terminal stem segment is A_(q−5).

Also provided herein are influenza hemagglutinin stem domainpolypeptides comprising deleted forms of HA1 C-terminal stem segmentswherein up to 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residues aredeleted from either or both termini of the HA1 C-terminal stem segment.In certain embodiments, provided herein are influenza hemagglutinin stemdomain polypeptides that comprise expanded forms of HA1 C-terminal stemsegments wherein 1, 2 or 3 residues are added to the N-terminus of theHA1 C-terminal stem segments; these added residues might be derived fromthe amino acid sequence of a globular head domain adjacent to an HA1C-terminal stem segment. In particular embodiments, if one residue isadded to the C-terminal stem segment, then one residue is added to theN-terminal stem segment; if two residues are added to the C-terminalstem segment, then two residues are added to the N-terminal stemsegment; if three residues are added to the C-terminal stem segment,then three residues are added to the N-terminal stem segment. Furtherprovided herein are influenza hemagglutinin stem domain polypeptidescomprising altered forms of HA1 C-terminal stem segments wherein up to10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residues are conservativelysubstituted with other amino acids. Further provided are influenzahemagglutinin stem domain polypeptides comprising deleted and alteredHA1 C-terminal stem segments.

The influenza hemagglutinin stem domain polypeptides might be based on(i.e. might have sequence identity, as described above) any influenzahemagglutinin known to those of skill or later discovered. In certainembodiments, influenza hemagglutinin stem domain polypeptides are basedon an influenza A hemagglutinin. In certain embodiments, the influenzahemagglutinin stem domain polypeptides are based on an influenza Ahemagglutinin selected from the group consisting of H1, H2, H3, H4, H5,H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 and H16. In certainembodiments, influenza hemagglutinin stem domain polypeptides are basedon an influenza B hemagglutinin, as described in detail below.

The HA1 N-terminal stem segments might be based on (i.e. might havesequence identity, as described above) any HA1 N-terminal stem segmentsknown to those of skill or later discovered. In certain embodiments, theHA1 N-terminal stem segments are based on influenza A HA1 N-terminalstem segments. In certain embodiments, the HA1 N-terminal stem segmentsare based on an influenza A hemagglutinin selected from the groupconsisting of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13,H14, H15 and H16. In certain embodiments, the HA1 N-terminal stemsegment is selected from SEQ ID NOS:34-49. In certain embodiments, theHA1 N-terminal stem segment is selected from SEQ ID NOS:34-49, eachhaving one amino acid deleted from its C-terminus. In certainembodiments, the HA1 N-terminal stem segment is selected from SEQ IDNOS:34-49, each having two amino acids deleted from its C-terminus. Incertain embodiments, the HA1 N-terminal stem segment is selected fromSEQ ID NOS:34-49, each having three amino acids deleted from itsC-terminus. In certain embodiments, the HA1 N-terminal stem segment isselected from SEQ ID NOS:34-49, each having four amino acids deletedfrom its C-terminus. In certain embodiments, the HA1 N-terminal stemsegment is selected from SEQ ID NOS:34-49, each having five amino acidsdeleted from its C-terminus. In certain embodiments, the HA1 N-terminalstem segment is selected from SEQ ID NOS:177-224.

The HA1 C-terminal stem segments might be based on (i.e. might havesequence identity, as described above) any HA1 C-terminal stem segmentsknown to those of skill or later discovered. In certain embodiments, theHA1 C-terminal stem segments are based on influenza A HA1 C-terminalstem segments. In certain embodiments, the HA1 C-terminal stem segmentsare based on an influenza A hemagglutinin selected from the groupconsisting of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13,H14, H15 and H16. In certain embodiments, the HA1 C-terminal stemsegment is selected from SEQ ID NOS:50-65. In certain embodiments, theHA1 C-terminal stem segment is selected from SEQ ID NOS: 50-65, eachhaving one amino acid deleted from its N-terminus. In certainembodiments, the HA1 C-terminal stem segment is selected from SEQ IDNOS: 50-65, each having two amino acids deleted from its N-terminus. Incertain embodiments, the HA1 C-terminal stem segment is selected fromSEQ ID NOS: 50-65, each having three amino acids deleted from itsN-terminus. In certain embodiments, the HA1 C-terminal stem segment isselected from SEQ ID NOS: 50-65, each having four amino acids deletedfrom its N-terminus. In certain embodiments, the HA1 C-terminal stemsegment is selected from SEQ ID NOS: 50-65, each having five amino acidsdeleted from its N-terminus. In certain embodiments, the HA1 C-terminalstem segment is selected from SEQ ID NOS:226-273.

The HA2 stem domains might be based on (i.e. might have sequenceidentity, as described above) any HA2 stem domains known to those ofskill or later discovered. In certain embodiments, the HA2 stem domainsare based on influenza A HA2 stem domains. In certain embodiments, theHA2 stem domains are based on an influenza A hemagglutinin selected fromthe group consisting of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11,H12, H13, H14, H15 and H16. In certain embodiments, the HA2 stem domainis selected from SEQ ID NOS:66-97.

In embodiments comprising a signal peptide, the signal peptide might bebased on any influenza virus signal peptide known to those of skill inthe art. In certain embodiments, the signal peptides are based oninfluenza A signal peptides. In certain embodiments, the signal peptidesare based on an influenza A hemagglutinin selected from the groupconsisting of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13,H14, H15 and H16. In certain embodiments, the signal peptide might beany signal peptide deemed useful to one of skill in the art. In certainembodiments, the signal peptide is selected from SEQ ID NOS:18-33.

In embodiments comprising a luminal domain, the luminal domain might bebased on any influenza luminal domain known to those of skill in theart. In certain embodiments, the luminal domains are based on influenzaA luminal domains. In certain embodiments, the HA2 luminal domains arebased on an influenza A hemagglutinin selected from the group consistingof H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 andH16. In certain embodiments, the luminal domain might be any luminaldomain deemed useful to one of skill in the art. In certain embodiments,the luminal domain is selected from SEQ ID NOS:98-113.

In embodiments comprising a transmembrane domain, the transmembranedomain might be based on any influenza transmembrane domain known tothose of skill in the art. In certain embodiments, the transmembranedomains are based on influenza A transmembrane domains. In certainembodiments, the HA2 transmembrane domains are based on an influenza Ahemagglutinin selected from the group consisting of H1, H2, H3, H4, H5,H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 and H16. In certainembodiments, the transmembrane domain might be any transmembrane domaindeemed useful to one of skill in the art. In certain embodiments, thetransmembrane domain is selected from SEQ ID NOS:114-129.

In embodiments comprising a cytoplasmic domain, the cytoplasmic domainmight be based on any influenza cytoplasmic domain known to those ofskill in the art. In certain embodiments, the cytoplasmic domains arebased on influenza A cytoplasmic domains. In certain embodiments, theHA2 cytoplasmic domains are based on an influenza A hemagglutininselected from the group consisting of H1, H2, H3, H4, H5, H6, H7, H8,H9, H10, H11, H12, H13, H14, H15 and H16. In certain embodiments, thecytoplasmic domain might be any cytoplasmic domain deemed useful to oneof skill in the art. In certain embodiments, the cytoplasmic domain isselected from SEQ ID NOS:130-145.

In certain embodiments, one or more of the glycosylation sites in thehemagglutinin stem domain are altered or deleted such that glycosylationat these sites will not occur during processing and maturation of thepolypeptide. Those of skill in the art will recognize that influenza HAtypically comprises one or more glycosylation sequences (e.g.Asn-Xaa-Ser/Thr/Cys, wherein Xaa is any amino acid other than Pro). Incertain embodiments, one or more amino acid residues in a glycosylationsequence is conservatively substituted with an amino acid residue thatdisrupts the glycosylation sequence. In certain embodiments, one or moreamino acid residues in a glycosylation sequence is substituted with anyamino acid residue that disrupts the glycosylation sequence. In certainembodiments, one or more asparagine residues in a glycosylation sequenceis substituted with alanine. In a particular embodiment, the asparagineat position 38 of an H3 hemagglutinin is changed to an alanine.

Table 1, below, identifies signal peptides, HA1 N-terminal stemsegments, HA1 C-terminal stem segments and HA2 domains of influenza Ahemagglutinin polypeptides. These signal peptides, stem segments anddomains are useful in the polypeptides and methods described herein.

TABLE 1 Exemplary Influenza A Hemagglutinin Sequences HA Subtype HA1 HA1(Genbank Signal N-terminal C-terminal No.) peptide Stem SegmentStem Segment HA2 Domain H1 MKAN DTICIGYHANN CNTKCQTPLG GLFGAIAGFIEGGWPR8-H1N1 LLVLL STDTVDTVLE AINSSLPYQNI TGMIDGWYGYHHQ (EF467821.1) CALAAKNVTVTHSVN HPVTIGECPKY NEQGSGYAADQKST ADA LLEDSHNGKL VRSAKLRMVTQNAINGITNKVNTVI [SEQ ID C GLRNNPSIQSR EKMNIQFTAVGKEF NO.: 18] [SEQ ID[SEQ ID NKLEKRMENLNKK NO.: 34] NO.: 50] VDDGFLDIWTYNAE LLVLLENERTLDFHDSNVKNLYEKVKSQ LKNNAKEIGNGCFE FYHKCDNECMESVR NGTYDYPKYSEESKLNREKVDGVKLES MGIYQILAIYSTVAS SLVLLVSLGAISFW MCSNGSLQCRICI[SEQ ID NO.: 66] H2 MAIIY DQICIGYHSNN CETKCQTPLG GLFGAIAGFIEGGW (L11136)LILLFT STEKVDTILER AINTTLPFHNV QGMIDGWYGYHHS AVRG NVTVTHAQNI HPLTIGECPKYNDQGSGYAADKEST [SEQ ID LEKTHNGKLC VKSERLVLAT QKAIDGITNRVNSVI NO.: 19][SEQ ID GLRNVPQIESR EKMNTQFEAVGKEF NO.: 35] [SEQ ID SNLEKRLENLNKKMNO.: 51] EDGFLDVWTYNAE LLVLMENERTLDFH DSNVKNLYDRVRM QLRDNAKELGNGCFEFYHKCDDECMNS VKNGTYDYPKYEEE SKLNRNEIKGVKLS NMGVYQILAIYATVAGSLSLAIMIAGISL WMCSNGSLQCRICI [SEQ ID NO.: 67] H3 MKTII QDLPGNDNSTCISECITPNGSI GLFGAIAGFIENGW HK68-H3N2 ALSYIF ATLCLGHHAV PNDKPFQNVNEGMIDGWYGFRHQ (EF409245) CLALG PNGTLVKTITD KITYGACPKY NSEGTGQAADLKSTPDB: 1HGJ [SEQ ID DQIEVTNATEL VKQNTLKLAT QAAIDQINGKLNRVI NO.: 20]VQSSSTGKIC GMRNVPEKQT EKTNEKFHQIEKEFS [SEQ ID R EVEGRIQDLEKYVE NO.: 36][SEQ ID DTKIDLWSYNAELL NO. 52] VALENQHTIDLTDS EMNKLFEKTRRQLRENAEDMGNGCFKIY HKCDNACIESIRNGT YDHDVYRDEALNN RFQIKGVELKSGYKDWILWISFAISCFLL CVVLLGFIMWACQR GNIRCNICI [SEQ ID NO.: 68] H4 MLSIVIQNYTGNPVIC CVSKCHTDKG GLFGAIAGFIENGW (D90302) LFLLIA MGHHAVANGSLSTTKPFQNI QGLIDGWYGFRHQ ENSS TMVKTLADDQ SRIAVGDCPRY NAEGTGTAADLKST[SEQ ID VEVVTAQELV VKQGSLKLAT QAAIDQINGKLNRLI NO.: 21] ESQNLPELCGMRNIPEKAS EKTNDKYHQIEKEF [SEQ ID R EQVEGRIQDLENYV NO.: 37] [SEQ IDEDTKIDLWSYNAEL NO.: 53] LVALENQHTIDVTD SEMNKLFERVRRQL RENAEDKGNGCFEIFHKCDNNCIESIRNG TYDHDIYRDEAINN RFQIQGVKLTQGYK DIILWISFSISCFLLVALLLAFILWACQNG NIRCQICI [SEQ ID NO.: 69] H5 MERIV DQICIGYHAN CDTKCQTPVGGLFGAIAGFIEGGW (X07826) LLLAI KSTKQVDTIM EINSSMPFHNI QGMVDGWYGYHH VSLVKEKNVTVTHAQ HPHTIGECPKY SNEQGSGYAADKES S DILERTHNGKL VKSDRLVLATTQKAIDGITNKVNSI [SEQ ID C GLRNVPQRKK IDKMNTRFEAVGKE NO.: 22] [SEQ ID RFNNLERRVENLNKK NO.: 38] [SEQ ID MEDGFLDVWTYNV NO.: 54] ELLVLMENERTLDFHDSNVNNLYDKVR LQLKDNARELGNGC FEFYHKCDNECMES VRNGTYDYPQYSEEARLNREEISGVKLES MGVYQILSIYSTVAS SLALAIMIAGLSFW MCSNGSLQCRICI[SEQ ID NO.: 70] H6 MIAIIV DKICIGYHAN CDATCQTVAG GLFGAIAGFIEGGW (D90303)VAILA NSTTQIDTILE VLRTNKTFQN TGMIDGWYGYHHE TAGRS KNVTVTHSVE VSPLWIGECPKNSQGSGYAADREST [SEQ ID LLENQKEERF YVKSESLRLA QKAVDGITNKVNSII NO.: 23] CTGLRNVPQIET DKMNTQFEAVDHE [SEQ ID R FSNLERRIDNLNKR NO.: 39] [SEQ IDMEDGFLDVWTYNA NO.: 55] ELLVLLENERTLDL HDANVKNLYERVK SQLRDNAMILGNGCFEFWHKCDDECMES VKNGTYDYPKYQD ESKLNRQEIESVKLE SLGVYQILAIYSTVSSSLVLVGLIIAVGLW MCSNGSMQCRICI [SEQ ID NO.: 71] H7 MNTQI DKICLGHHAVCEGECYHSGG GLFGAIAGFIENGW (M24457) LVFAL SNGTKVNTLT TITSRLPFQNINEGLVDGWYGFRHQ VAVIP ERGVEVVNAT SRAVGKCPRY NAQGEGTAADYKS TNA ETVERTNIPKIVKQESLLLAT TQSAIDQITGKLNRL [SEQ ID C GMKNVPEPSK IEKTNQQFELIDNEF NO.: 24][SEQ ID KRKKR TEVEKQIGNLINWT NO.: 40] [SEQ ID KDSITEVWSYNAELI NO.: 56]VAMENQHTIDLADS EMNRLYERVRKQL RENAEEDGTGCFEIF HKCDDDCMASIRNNTYDHSKYREEAMQ NRIQIDPVKLSSGYK DVILWFSFGASCFLL LAIAMGLVFICVKN GNMRCTICI[SEQ ID NO.: 72] H8 MEKFI DRICIGYQSNN CNTKCQTYAG GLFGAIAGFIEGGWS(D90304) AIATL STDTVNTLIEQ AINSSKPFQNA GMIDGWYGFHHSN ASTNA NVPVTQTMELSRHYMGECPK SEGTGMAADQKST Y VETEKHPAYC YVKKASLRLA QEAIDKITNKVNNIV [SEQ ID[SEQ ID VGLRNTPSVEP DKMNREFEVVNHEF NO.: 25] NO.: 41] R SEVEKRINMINDKID[SEQ ID DQIEDLWAYNAELL NO.: 57] VLLENQKTLDEHDS NVKNLFDEVKRRLSANAIDAGNGCFDIL HKCDNECMETIKNG TYDHKEYEEEAKLE RSKINGVKLEENTTYKILSIYSTVAASLC LAILIAGGLILGMQN GSCRCMFCI [SEQ ID NO.: 73] H9 METKDKICIGYQSTN CVVQCQTEKG GLFGAIAGFIEGGWP (D90305) AIIAAL STETVDTLTESGLNTTLPFHNI GLVAGWYGFQHSN LMVTA NVPVTHTKEL SKYAFGNCPK DQGVGMAADKGST ANALHTEHNGMLC YVGVKSLKLP QKAIDKITSKVNNII [SEQ ID [SEQ ID VGLRNVPAVSDKMNKQYEVIDHEF NO.: 26] NO.: 42] SR NELEARLNMINNKI [SEQ IDDDQIQDIWAYNAEL NO.: 58] LVLLENQKTLDEHD ANVNNLYNKVKRA LGSNAVEDGNGCFELYHKCDDQCMETIR NGTYDRQKYQEESR LERQKIEGVKLESEG TYKILTIYSTVASSLVLAMGFAAFLFWA MSNGSCRCNICI [SEQ ID NO.: 74] H10 MYKV LDRICLGHHACESKCFWRGG GLFGAIAGFIENGW (M21647) VVIIAL VANGTIVKTL SINTKLPFQNLEGMVDGWYGFRHQ LGAVK TNEQEEVTNA SPRTVGQCPK NAQGTGQAADYKS G TETVESTNLNYVNQRSLLLA TQAAIDQITGKLNRL [SEQ ID KLC TGMRNVPEVV IEKTNTEFESIESEFSNO.: 27] [SEQ ID QGR ETEHQIGNVINWTK NO.: 43] [SEQ ID DSITDIWTYNAELLVNO.: 59] AMENQHTIDMADSE MLNLYERVRKQLR QNAEEDGKGCFEIY HTCDDSCMESIRNNTYDHSQYREEALLN RLNINPVKLSSGYK DIILWFSFGESCFVL LAVVMGLVFFCLKN GNMRCTICI[SEQ ID NO.: 75] H11 MEKTL DEICIGYLSNN CSTKCQTEIGG GLFGAIAGFIEGGWP(D90306) LFAAIF STDKVDTIIEN INTNKSFHNV GLINGWYGFQHRDE LCVKA NVTVTSSVELHRNTIGDCPK EGTGIAADKESTQK [SEQ ID VETEHTGSFC YVNVKSLKLA AIDQITSKVNNIVDRNO.: 28] [SEQ ID TGPRNVPAIAS MNTNFESVQHEFSEI NO.: 44] R EERINQLSKHVDDS[SEQ ID VVDIWSYNAQLLVL NO.: 60] LENEKTLDLHDSNV RNLHEKVRRMLKDNAKDEGNGCFTFYH KCDNKCIERVRNGT YDHKEFEEESKINR QEIEGVKLDSSGNVYKILSIYSCIASSLVL AALIMGFMFWACS NGSCRCTICI [SEQ ID NO.: 76] H12 MEKFIIDKICIGYQTNN CVTECQLNEG GLFGAIAGFIEGGWP (D90307) LSTVL STETVNTLSEQVMNTSKPFQN GLVAGWYGFQHQN AASFA NVPVTQVEEL TSKHYIGKCPK AEGTGIAADRDSTQ YVHRGIDPILC YIPSGSLKLAI RAIDNMQNKLNNVI [SEQ ID [SEQ ID GLRNVPQVQDDKMNKQFEVVNHE NO.: 29] NO.: 45] R FSEVESRINMINSKI [SEQ ID DDQITDIWAYNAELNO.: 61] LVLLENQKTLDEHD ANVRNLHDRVRRV LRENAIDTGDGCFEI LHKCDNNCMDTIRNGTYNHKEYEEESKI ERQKVNGVKLEENS TYKILSIYSSVASSL VLLLMIIGGFIFGCQ NGNVRCTFCI[SEQ ID NO.: 77] H13 MALN DRICVGYLSTN CNTKCQTSVG GLFGAIAGFIEGGWP(D90308) VIATL SSERVDTLLEN GINTNRTFQNI GLINGWYGFQHQNE TLISVCGVPVTSSIDLIE DKNALGDCPK QGTGIAADKESTQK VHA TNHTGTYC YIKSGQLKLATAIDQITTKINNIIDKM [SEQ ID [SEQ ID GLRNVPAISNR NGNYDSIRGEFNQV NO.: 30]NO.: 46] [SEQ ID EKRINMLADRIDDA NO.: 62] VTDIWSYNAKLLVL LENDKTLDMHDANVKNLHEQVRRELKD NAIDEGNGCFELLH KCNDSCMETIRNGT YDHTEYAEESKLKRQEIDGIKLKSEDNVY KALSIYSCIASSVVL VGLILSFIMWACSSG NCRFNVCI[SEQ ID NO.: 78] H14 MIALIL QITNGTTGNPII CTSPCLTDKGS GLFGAIAGFIENGW(M35997) VALAL CLGHHAVENG IQSDKPFQNVS QGLIDGWYGFRHQ SHTAY TSVKTLTDNHRIAIGNCPKYV NAEGTGTAADLKST S VEVVSAKELV KQGSLMLATG QAAIDQINGKLNRLI[SEQ ID ETNHTDELC MRNIPGKQAK EKTNEKYHQIEKEF NO.: 31] [SEQ ID [SEQ IDEQVEGRIQDLEKYV NO.: 47] NO.: 63] EDTKIDLWSYNAEL LVALENQHTIDVTDSEMNKLFERVRRQL RENAEDQGNGCFEI FHQCDNNCIESIRNG TYDHNIYRDEAINNRIKINPVTLTMGYK DIILWISFSMSCFVF VALILGFVLWACQN GNIRCQICI [SEQ ID NO.: 79]H15 MNTQI DKICLGHHAV CEGECFYSGG GLFGAIAGFIENGW (L43917) IVILVLANGTKVNTLT TINSPLPFQNID EGLIDGWYGFRHQN GLSMV ERGVEVVNAT SRAVGKCPRYAQGQGTAADYKST KS ETVEITGIDKV VKQSSLPLAL QAAIDQITGKLNRLI [SEQ ID CGMKNVPEKIR EKTNKQFELIDNEFT NO.: 32] [SEQ ID TR EVEQQIGNVINWTR NO.: 48][SEQ ID DSLTEIWSYNAELL NO.: 64] VAMENQHTIDLADS EMNKLYERVRRQLRENAEEDGTGCFEIF HRCDDQCMESIRNN TYNHTEYRQEALQN RIMINPVKLSSGYKDVILWFSFGASCVML LAIAMGLIFMCVKN GNLRCTICI [SEQ ID NO.: 80] H16 MMIKDKICIGYLSNN CNTKCQTSLG GLFGAIAGFIEGGWP (EU293865) VLYFLI SSDTVDTLTENGINTNKTFQNI GLINGWYGFQHQNE IVLGR GVPVTSSVDL ERNALGDCPK QGTGIAADKASTQKYSKA VETNHTGTYC YIKSGQLKLAT AINEITTKINNIIEKM [SEQ ID [SEQ ID GLRNVPSIGERNGNYDSIRGEFNQV NO.: 33] NO.: 49] [SEQ ID EKRINMLADRVDDA NO.: 65]VTDIWSYNAKLLVL LENDRTLDLHDANV RNLHDQVKRALKS NAIDEGDGCFNLLHKCNDSCMETIRNGT YNHEDYREESQLKR QEIEGIKLKTEDNVY KVLSIYSCIASSIVLVGLILAFIMWACSNG SCRFNVCI [SEQ ID NO.: 81]

Table 1A, below, identifies useful HA1 N-terminal stem segments and HA1C-terminal stem segments for the polypeptides and methods describedherein.

TABLE 1A Exemplary Influenza A Hemagglutinin Sequences HA Subtype(Genbank HA1 N-terminal HA1 No.) Stem Segment C-terminal Stem Segment H1DTICIGYHANNSTDTVDT NTKCQTPLGAINSSLPYQNIHPVTIGEC PR8-H1N1VLEKNVTVTHSVNLLED PKYVRSAKLRMVTGLRNNPSIQSR (EF467821.1) SHNGKL[SEQ ID NO.: 226] No Cys [SEQ ID NO.: 177] H1 DTICIGYHANNSTDTVDTTKCQTPLGAINSSLPYQNIHPVTIGECP PR8-H1N1 VLEKNVTVTHSVNLLEDKYVRSAKLRMVTGLRNNPSIQSR (EF467821.1) SHNGKL [SEQ ID NO.: 227] No Cys Δ1[SEQ ID NO.: 178] H1 DTICIGYHANNSTDTVDT KCQTPLGAINSSLPYQNIHPVTIGECPKPR8-H1N1 VLEKNVTVTHSVNLLED YVRSAKLRMVTGLRNNPSIQSR (EF467821.1) SHNGK[SEQ ID NO.: 228] No Cys Δ3 [SEQ ID NO.: 179] H1 DTICIGYHANNSTDTVDTCKCQTPLGAINSSLPYQNIHPVTIGECP PR8-H1N1 VLEKNVTVTHSVNLLEDKYVRSAKLRMVTGLRNNPSIQSRG (EF467821.1) SHNGKLCRLKC [SEQ ID NO.: 310]PR8-CON-A [SEQ ID NO.: 309] H2 DQICIGYHSNNSTEKVDTETKCQTPLGAINTTLPFHNVHPLTIGE (L11136) ILERNVTVTHAQNILEKTCPKYVKSERLVLATGLRNVPQIESR No Cys HNGKL [SEQ ID NO.: 229][SEQ ID NO.: 180] H2 DQICIGYHSNNSTEKVDT TKCQTPLGAINTTLPFHNVHPLTIGECP(L11136) ILERNVTVTHAQNILEKT KYVKSERLVLATGLRNVPQIESR No Cys Δ1 HNGKL[SEQ ID NO.: 230] [SEQ ID NO.: 181] H2 DQICIGYHSNNSTEKVDTKCQTPLGAINTTLPFHNVHPLTIGECP (L11136) ILERNVTVTHAQNILEKTKYVKSERLVLATGLRNVPQIESR No Cys Δ3 HNGK [SEQ ID NO.: 231][SEQ ID NO.: 182] H3 QDLPGNDNSTATLCLGH ISECITPNGSIPNDKPFQNVNKITYGACHK68-H3N2 HAVPNGTLVKTITDDQIE PKYVKQNTLKLATGMRNVPEKQTR (EF409245)VTNATELVQSSSTGKI [SEQ ID NO.: 232] PDB: 1HGJ [SEQ ID NO.: 183] No Cys H3QDLPGNDNSTATLCLGH SECITPNGSIPNDKPFQNVNKITYGACP HK68-H3N2HAVPNGTLVKTITDDQIE KYVKQNTLKLATGMRNVPEKQTR (EF409245) VTNATELVQSSSTGKI[SEQ ID NO.: 233] PDB: 1HGJ [SEQ ID NO.: 184] No Cys Δ1 H3QDLPGNDNSTATLCLGH ECITPNGSIPNDKPFQNVNKITYGACP HK68-H3N2HAVPNGTLVKTITDDQIE KYVKQNTLKLATGMRNVPEKQTR (EF409245) VTNATELVQSSSTGK[SEQ ID NO.: 234] PDB: 1HGJ [SEQ ID NO.: 185] No Cys Δ3 H3STATLCLGHHAVPNGTL CISECITPNGSIPNDKPFQNVNKITYGA HK68-H3N2VKTITDDQIEVTNATELV CPKYVKQNTLKLATGMRNVPEKQTR PDB: 1HGJ QSSSTGKIC[SEQ ID NO.: 52] (EF409245) [SEQ ID NO.: 308] HK68-CON-A H4QNYTGNPVICMGHHAV VSKCHTDKGSLSTTKPFQNISRIAVGD (D90302) ANGTMVKTLADDQVEVCPRYVKQGSLKLATGMRNIPEKASR No Cys VTAQELVESQNLPEL [SEQ ID NO.: 235][SEQ ID NO.: 186] H4 QNYTGNPVICMGHHAV SKCHTDKGSLSTTKPFQNISRIAVGDC(D90302) ANGTMVKTLADDQVEV PRYVKQGSLKLATGMRNIPEKASR No Cys Δ1VTAQELVESQNLPEL [SEQ ID NO.: 236] [SEQ ID NO.: 187] H4 QNYTGNPVICMGHHAVKCHTDKGSLSTTKPFQNISRIAVGDCP (D90302) ANGTMVKTLADDQVEVRYVKQGSLKLATGMRNIPEKASR No Cys Δ3 VTAQELVESQNLPE [SEQ ID NO.: 237][SEQ ID NO.: 188] H5 DQICIGYHANKSTKQVD DTKCQTPVGEINSSMPFHNIHPHTIGE(X07826) TIMEKNVTVTHAQDILE CPKYVKSDRLVLATGLRNVPQRKKR No Cys RTHNGKL[SEQ ID NO.: 238] [SEQ ID NO.: 189] H5 DQICIGYHANKSTKQVDTKCQTPVGEINSSMPFHNIHPHTIGECP (X07826) TIMEKNVTVTHAQDILEKYVKSDRLVLATGLRNVPQRKKR No Cys Δ1 RTHNGKL [SEQ ID NO.: 239][SEQ ID NO.: 190] H5 DQICIGYHANKSTKQVD KCQTPVGEINSSMPFHNIHPHTIGECPK(X07826) TIMEKNVTVTHAQDILE YVKSDRLVLATGLRNVPQRKKR No Cys Δ3 RTHNGK[SEQ ID NO.: 240] [SEQ ID NO.: 191] H6 DKICIGYHANNSTTQIDTDATCQTVAGVLRTNKTFQNVSPLWIG (D90303) ILEKNVTVTHSVELLENQECPKYVKSESLRLATGLRNVPQIETR No Cys KEERF [SEQ ID NO.: 241][SEQ ID NO.: 192] H6 DKICIGYHANNSTTQIDT ATCQTVAGVLRTNKTFQNVSPLWIGE(D90303) ILEKNVTVTHSVELLENQ CPKYVKSESLRLATGLRNVPQIETR No Cys Δ1 KEERF[SEQ ID NO.: 242] [SEQ ID NO.: 193] H6 DKICIGYHANNSTTQIDTTCQTVAGVLRTNKTFQNVSPLWIGEC (D90303) ILEKNVTVTHSVELLENQPKYVKSESLRLATGLRNVPQIETR No Cys Δ3 KEER [SEQ ID NO.: 243][SEQ ID NO.: 194] H7 DKICLGHHAVSNGTKVN EGECYHSGGTITSRLPFQNINSRAVGK(M24457) TLTERGVEVVNATETVE CPRYVKQESLLLATGMKNVPEPSKKR No Cys RTNIPKI KKR[SEQ ID NO.: 195] [SEQ ID NO.: 244] H7 DKICLGHHAVSNGTKVNGECYHSGGTITSRLPFQNINSRAVGKC (M24457) TLTERGVEVVNATETVEPRYVKQESLLLATGMKNVPEPSKKRK No Cys Δ1 RTNIPKI KR [SEQ ID NO.: 196][SEQ ID NO.: 245] H7 DKICLGHHAVSNGTKVN ECYHSGGTITSRLPFQNINSRAVGKCP(M24457) TLTERGVEVVNATETVE RYVKQESLLLATGMKNVPEPSKKRKK No Cys Δ3 RTNIPK R[SEQ ID NO.: 197] [SEQ ID NO.: 246] H8 DRICIGYQSNNSTDTVNTNTKCQTYAGAINSSKPFQNASRHYMG (D90304) LIEQNVPVTQTMELVETECPKYVKKASLRLAVGLRNTPSVEPR No Cys EKHPAY [SEQ ID NO.: 247][SEQ ID NO.: 198] H8 DRICIGYQSNNSTDTVNT TKCQTYAGAINSSKPFQNASRHYMGE(D90304) LIEQNVPVTQTMELVET CPKYVKKASLRLAVGLRNTPSVEPR No Cys Δ1 EKHPAY[SEQ ID NO.: 248] [SEQ ID NO.: 199] H8 DRICIGYQSNNSTDTVNTKCQTYAGAINSSKPFQNASRHYMGEC (D90304) LIEQNVPVTQTMELVETPKYVKKASLRLAVGLRNTPSVEPR No Cys Δ3 EKHPA [SEQ ID NO.: 249][SEQ ID NO.: 200] H9 DKICIGYQSTNSTETVDT VVQCQTEKGGLNTTLPFHNISKYAFG(D90305) LTESNVPVTHTKELLHTE NCPKYVGVKSLKLPVGLRNVPAVSSR No Cys HNGML[SEQ ID NO.: 250] [SEQ ID NO.: 201] H9 DKICIGYQSTNSTETVDTVQCQTEKGGLNTTLPFHNISKYAFGN (D90305) LTESNVPVTHTKELLHTECPKYVGVKSLKLPVGLRNVPAVSSR No Cys Δ1 HNGML [SEQ ID NO.: 251][SEQ ID NO.: 202] H9 DKICIGYQSTNSTETVDT QCQTEKGGLNTTLPFHNISKYAFGNCP(D90305) LTESNVPVTHTKELLHTE KYVGVKSLKLPVGLRNVPAVSSR No Cys Δ3 HNGM[SEQ ID NO.: 252] [SEQ ID NO.: 203] H10 LDRICLGHHAVANGTIVESKCFWRGGSINTKLPFQNLSPRTVGQ (M21647) KTLTNEQEEVTNATETVCPKYVNQRSLLLATGMRNVPEVVQG No Cys ESTNLNKL R [SEQ ID NO.: 204][SEQ ID NO.: 253] H10 LDRICLGHHAVANGTIV SKCFWRGGSINTKLPFQNLSPRTVGQC(M21647) KTLTNEQEEVTNATETV PKYVNQRSLLLATGMRNVPEVVQGR No Cys Δ1 ESTNLNKL[SEQ ID NO.: 254] [SEQ ID NO.: 205] H10 LDRICLGHHAVANGTIVKCFWRGGSINTKLPFQNLSPRTVGQCP (M21647) KTLTNEQEEVTNATETVKYVNQRSLLLATGMRNVPEVVQGR No Cys Δ3 ESTNLNK [SEQ ID NO.: 255][SEQ ID NO.: 206] H11 DEICIGYLSNNSTDKVDT STKCQTEIGGINTNKSFHNVHRNTIGD(D90306) IIENNVTVTSSVELVETE CPKYVNVKSLKLATGPRNVPAIASR No Cys HTGSF[SEQ ID NO.: 256] [SEQ ID NO.: 207] H11 DEICIGYLSNNSTDKVDTTKCQTEIGGINTNKSFHNVHRNTIGDC (D90306) IIENNVTVTSSVELVETEPKYVNVKSLKLATGPRNVPAIASR No Cys Δ1 HTGSF [SEQ ID NO.: 257][SEQ ID NO.: 208] H11 DEICIGYLSNNSTDKVDT KCQTEIGGINTNKSFHNVHRNTIGDCP(D90306) IIENNVTVTSSVELVETE KYVNVKSLKLATGPRNVPAIASR No Cys Δ3 HTGS[SEQ ID NO.: 258] [SEQ ID NO.: 209] H12 DKICIGYQTNNSTETVNTVTECQLNEGVMNTSKPFQNTSKHYIG (D90307) LSEQNVPVTQVEELVHRKCPKYIPSGSLKLAIGLRNVPQVQDR No Cys GIDPIL [SEQ ID NO.: 259][SEQ ID NO.: 210] H12 DKICIGYQTNNSTETVNT TECQLNEGVMNTSKPFQNTSKHYIGK(D90307) LSEQNVPVTQVEELVHR CPKYIPSGSLKLAIGLRNVPQVQDR No Cys Δ1 GIDPIL[SEQ ID NO.: 260] [SEQ ID NO.: 211] H12 DKICIGYQTNNSTETVNTECQLNEGVMNTSKPFQNTSKHYIGKC (D90307) LSEQNVPVTQVEELVHRPKYIPSGSLKLAIGLRNVPQVQDR No Cys Δ3 GIDPI [SEQ ID NO.: 261][SEQ ID NO.: 212] H13 DRICVGYLSTNSSERVDT NTKCQTSVGGINTNRTFQNIDKNALG(D90308) LLENGVPVTSSIDLIETN DCPKYIKSGQLKLATGLRNVPAISNR No Cys HTGTY[SEQ ID NO.: 262] [SEQ ID NO.: 213] H13 DRICVGYLSTNSSERVDTTKCQTSVGGINTNRTFQNIDKNALGD (D90308) LLENGVPVTSSIDLIETNCPKYIKSGQLKLATGLRNVPAISNR No Cys Δ1 HTGTY [SEQ ID NO.: 263][SEQ ID NO.: 214] H13 DRICVGYLSTNSSERVDT KCQTSVGGINTNRTFQNIDKNALGDC(D90308) LLENGVPVTSSIDLIETN PKYIKSGQLKLATGLRNVPAISNR No Cys Δ3 HTGT[SEQ ID NO.: 264] [SEQ ID NO.: 215] H14 QITNGTTGNPIICLGHHATSPCLTDKGSIQSDKPFQNVSRIAIGNC (M35997) VENGTSVKTLTDNHVEVPKYVKQGSLMLATGMRNIPGKQAK No Cys VSAKELVETNHTDEL [SEQ ID NO.: 265][SEQ ID NO.: 216] H14 QITNGTTGNPIICLGHHA SPCLTDKGSIQSDKPFQNVSRIAIGNCP(M35997) VENGTSVKTLTDNHVEV KYVKQGSLMLATGMRNIPGKQAK No Cys Δ1VSAKELVETNHTDEL [SEQ ID NO.: 266] [SEQ ID NO.: 217] H14QITNGTTGNPIICLGHHA PCLTDKGSIQSDKPFQNVSRIAIGNCPK (M35997)VENGTSVKTLTDNHVEV YVKQGSLMLATGMRNIPGKQAK No Cys Δ3 VSAKELVETNHTDE[SEQ ID NO.: 267] [SEQ ID NO.: 218] H15 DKICLGHHAVANGTKVEGECFYSGGTINSPLPFQNIDSRAVGK (L43917) NTLTERGVEVVNATETVCPRYVKQSSLPLALGMKNVPEKIRTR No Cys EITGIDKV [SEQ ID NO.: 268][SEQ ID NO.: 219] H15 DKICLGHHAVANGTKV GECFYSGGTINSPLPFQNIDSRAVGKC(L43917) NTLTERGVEVVNATETV PRYVKQSSLPLALGMKNVPEKIRTR No Cys Δ1 EITGIDKV[SEQ ID NO.: 269] [SEQ ID NO.: 220] H15 DKICLGHHAVANGTKVECFYSGGTINSPLPFQNIDSRAVGKCP (L43917) NTLTERGVEVVNATETVRYVKQSSLPLALGMKNVPEKIRTR No Cys Δ3 EITGIDK [SEQ ID NO.: 270][SEQ ID NO.: 221] H16 DKICIGYLSNNSSDTVDT NTKCQTSLGGINTNKTFQNIERNALGD(EU293865) LTENGVPVTSSVDLVET CPKYIKSGQLKLATGLRNVPSIGER No Cys NHTGTY[SEQ ID NO.: 271] [SEQ ID NO.: 222] H16 DKICIGYLSNNSSDTVDTTKCQTSLGGINTNKTFQNIERNALGDC (EU293865) LTENGVPVTSSVDLVETPKYIKSGQLKLATGLRNVPSIGER No Cys Δ1 NHTGTY [SEQ ID NO.: 272][SEQ ID NO.: 223] H16 DKICIGYLSNNSSDTVDT KCQTSLGGINTNKTFQNIERNALGDCP(EU293865) LTENGVPVTSSVDLVET KYIKSGQLKLATGLRNVPSIGER No Cys Δ3 NHTGT[SEQ ID NO.: 273] [SEQ ID NO.: 224]

Table 2, below, identifies putative stem domains, luminal domains,transmembrane domains and cytoplasmic domains of HA2 polypeptides.

TABLE 2 Exemplary Influenza A Hemagglutinin Sequences HA2 domain Subtype(Genbank Luminal Transmembrane Cytoplasmic No.) Stem domain domaindomain domain H1 GLFGAIAGFIEGGWT MGIYQ ILAIYSTVASSL NGSLQCRI PR8-H1N1GMIDGWYGYHHQNE [SEQ ID VLLVSLGAISF CI (EF467821.1) QGSGYAADQKSTQNNO.: 98] WMCS [SEQ ID AINGITNKVNTVIEK [SEQ ID NO.: 130] MNIQFTAVGKEFNKLNO.: 114] EKRMENLNKKVDDG FLDIWTYNAELLVLL ENERTLDFHDSNVKN LYEKVKSQLKNNAKEIGNGCFEFYHKCDN ECMESVRNGTYDYP KYSEESKLNREKVDG VKLES [SEQ ID NO.: 82] H2GLFGAIAGFIEGGWQ MGVYQ ILAIYATVAGSL NGSLQCRI (L11136) GMIDGWYGYHHSND[SEQ ID SLAIMIAGISLW CI QGSGYAADKESTQK NO.: 99] MCS [SEQ IDAIDGITNRVNSVIEK [SEQ ID NO.: 131] MNTQFEAVGKEFSNL NO.: 115]EKRLENLNKKMEDG FLDVWTYNAELLVL MENERTLDFHDSNV KNLYDRVRMQLRDNAKELGNGCFEFYHKC DDECMNSVKNGTYD YPKYEEESKLNRNEI KGVKLSN [SEQ ID NO.: 83]H3 GLFGAIAGFIENGWE SGYKD WILWISFAISCF RGNIRCNI HK68-H3N2 GMIDGWYGFRHQNS[SEQ ID LLCVVLLGFIM CI (EF409245) EGTGQAADLKSTQA NO.: 100] WACQ [SEQ IDPDB: 1HGJ AIDQINGKLNRVIEKT [SEQ ID NO.: 132] NEKFHQIEKEFSEVE NO.: 116]GRIQDLEKYVEDTKI DLWSYNAELLVALE NQHTIDLTDSEMNKL FEKTRRQLRENAEDMGNGCFKIYHKCDN ACIESIRNGTYDHDV YRDEALNNRFQIKGV ELK [SEQ ID NO.: 84] H4GLFGAIAGFIENGWQ QGYKD IILWISFSISCFLL NGNIRCQI (D90302) GLIDGWYGFRHQNA[SEQ ID VALLLAFILWA CI EGTGTAADLKSTQA NO.: 101] CQ [SEQ IDAIDQINGKLNRLIEKT [SEQ ID NO.: 133] NDKYHQIEKEFEQVE NO.: 117]GRIQDLENYVEDTKI DLWSYNAELLVALE NQHTIDVTDSEMNKL FERVRRQLRENAEDKGNGCFEIFHKCDNNC IESIRNGTYDHDIYRD EAINNRFQIQGVKLT [SEQ ID NO.: 85] H5GLFGAIAGFIEGGWQ MGVYQ ILSIYSTVASSL NGSLQCRI (X07826) GMVDGWYGYHHSN[SEQ ID ALAIMIAGLSF CI EQGSGYAADKESTQ NO.: 102] WMCS [SEQ IDKAIDGITNKVNSIIDK [SEQ ID NO.: 134] MNTRFEAVGKEFNN NO.: 118]LERRVENLNKKMED GFLDVWTYNVELLV LMENERTLDFHDSNV NNLYDKVRLQLKDNARELGNGCFEFYHKC DNECMESVRNGTYD YPQYSEEARLNREEIS GVKLES [SEQ ID NO.: 86]H6 GLFGAIAGFIEGGWT LGVYQ ILAIYSTVSSSL NGSMQCR (D90303) GMIDGWYGYHHENS[SEQ ID VLVGLIIAVGL ICI QGSGYAADRESTQK NO.: 103] WMCS [SEQ IDAVDGITNKVNSIIDK [SEQ ID NO.: 135] MNTQFEAVDHEFSNL NO.: 119]ERRIDNLNKRMEDGF LDVWTYNAELLVLL ENERTLDLHDANVK NLYERVKSQLRDNAMILGNGCFEFWHKC DDECMESVKNGTYD YPKYQDESKLNRQEI ESVKLES [SEQ ID NO.: 87]H7 GLFGAIAGFIENGWE SGYKD VILWFSFGASCF NGNMRCT (M24457) GLVDGWYGFRHQNA[SEQ ID LLLAIAMGLVFI ICI QGEGTAADYKSTQS NO.: 104] CVK [SEQ IDAIDQITGKLNRLIEKT [SEQ ID NO.: 136] NQQFELIDNEFTEVE NO.: 120]KQIGNLINWTKDSITE VWSYNAELIVAMEN QHTIDLADSEMNRLY ERVRKQLRENAEEDGTGCFEIFHKCDDDC MASIRNNTYDHSKYR EEAMQNRIQIDPVKL S [SEQ ID NO.: 88] H8GLFGAIAGFIEGGWS NTTYK ILSIYSTVAASL NGSCRCM (D90304) GMIDGWYGFHHSNS[SEQ ID CLAILIAGGLIL FCI EGTGMAADQKSTQE NO.: 105] GMQ [SEQ IDAIDKITNKVNNIVDK [SEQ ID NO.: 137] MNREFEVVNHEFSEV NO.: 121]EKRINMINDKIDDQIE DLWAYNAELLVLLE NQKTLDEHDSNVKN LFDEVKRRLSANAIDAGNGCFDILHKCDNE CMETIKNGTYDHKE YEEEAKLERSKINGV KLEE [SEQ ID NO.: 89] H9GLFGAIAGFIEGGWP EGTYK ILTIYSTVASSL NGSCRCNI (D90305) GLVAGWYGFQHSND[SEQ ID VLAMGFAAFLF CI QGVGMAADKGSTQK NO.: 106] WAMS [SEQ IDAIDKITSKVNNIIDKM [SEQ ID NO.: 138] NKQYEVIDHEFNELE NO.: 122]ARLNMINNKIDDQIQ DIWAYNAELLVLLEN QKTLDEHDANVNNL YNKVKRALGSNAVEDGNGCFELYHKCDD QCMETIRNGTYDRQ KYQEESRLERQKIEG VKLES [SEQ ID NO.: 90] H10GLFGAIAGFIENGWE SGYKD IILWFSFGESCF NGNMRCT (M21647) GMVDGWYGFRHQN[SEQ ID VLLAVVMGLV ICI AQGTGQAADYKSTQ NO.: 107] FFCLK [SEQ IDAAIDQITGKLNRLIEK [SEQ ID NO.: 139] TNTEFESIESEFSETEH NO.: 123]QIGNVINWTKDSITDI WTYNAELLVAMENQ HTIDMADSEMLNLYE RVRKQLRQNAEEDGKGCFEIYHTCDDSCM ESIRNNTYDHSQYRE EALLNRLNINPVKLS [SEQ ID NO.: 91] H11GLFGAIAGFIEGGWP GNVYK ILSIYSCIASSLV NGSCRCTI (D90306) GLINGWYGFQHRDE[SEQ ID LAALIMGFMFW CI EGTGIAADKESTQKA NO.: 108] ACS [SEQ IDIDQITSKVNNIVDRM [SEQ ID NO.: 140] NTNFESVQHEFSEIEE NO.: 124]RINQLSKHVDDSVVD IWSYNAQLLVLLENE KTLDLHDSNVRNLHE KVRRMLKDNAKDEGNGCFTFYHKCDNKCI ERVRNGTYDHKEFEE ESKINRQEIEGVKLDS S [SEQ ID NO.: 92] H12GLFGAIAGFIEGGWP NSTYK ILSIYSSVASSLV GNVRCTF (D90307) GLVAGWYGFQHQNA[SEQ ID LLLMIIGGFIFG CI EGTGIAADRDSTQRA NO.: 109] CQN [SEQ IDIDNMQNKLNNVIDK [SEQ ID NO.: 141] MNKQFEVVNHEFSE NO.: 125]VESRINMINSKIDDQI TDIWAYNAELLVLLE NQKTLDEHDANVRN LHDRVRRVLRENAIDTGDGCFEILHKCDNN CMDTIRNGTYNHKE YEEESKIERQKVNGV KLEE [SEQ ID NO.: 93] H13GLFGAIAGFIEGGWP DNVYK ALSIYSCIASSV GNCRFNV (D90308) GLINGWYGFQHQNE[SEQ ID VLVGLILSFIM CI QGTGIAADKESTQKA NO.: 110] WACSS [SEQ IDIDQITTKINNIIDKMN [SEQ ID NO.: 142] GNYDSIRGEFNQVEK NO.: 126]RINMLADRIDDAVTD IWSYNAKLLVLLEND KTLDMHDANVKNLH EQVRRELKDNAIDEGNGCFELLHKCNDSC METIRNGTYDHTEYA EESKLKRQEIDGIKLK SE [SEQ ID NO.: 94] H14GLFGAIAGFIENGWQ MGYKD IILWISFSMSCF NGNIRCQI (M35997) GLIDGWYGFRHQNA[SEQ ID VFVALILGFVL CI EGTGTAADLKSTQA NO.: 111] WACQ [SEQ IDAIDQINGKLNRLIEKT [SEQ ID NO.: 143] NEKYHQIEKEFEQVE NO.: 127]GRIQDLEKYVEDTKI DLWSYNAELLVALE NQHTIDVTDSEMNKL FERVRRQLRENAEDQGNGCFEIFHQCDNNC IESIRNGTYDHNIYRD EAINNRIKINPVTLT [SEQ ID NO.: 95] H15GLFGAIAGFIENGWE SGYKD VILWFSFGASC GNLRCTIC (L43917) GLIDGWYGFRHQNA[SEQ ID VMLLAIAMGLI I QGQGTAADYKSTQA NO.: 112] FMCVKN [SEQ IDAIDQITGKLNRLIEKT [SEQ ID NO.: 144] NKQFELIDNEFTEVE NO.: 128]QQIGNVINWTRDSLT EIWSYNAELLVAME NQHTIDLADSEMNKL YERVRRQLRENAEEDGTGCFEIFHRCDDQC MESIRNNTYNHTEYR QEALQNRIMINPVKL S [SEQ ID NO.: 96] H16GLFGAIAGFIEGGWP DNVYK VLSIYSCIASSIV NGSCRFN (EU293865) GLINGWYGFQHQNE[SEQ ID LVGLILAFIMW VCI QGTGIAADKASTQKA NO.: 113] ACS [SEQ IDINEITTKINNIIEKMNG [SEQ ID NO.: 145] NYDSIRGEFNQVEKR NO.: 129]INMLADRVDDAVTDI WSYNAKLLVLLEND RTLDLHDANVRNLH DQVKRALKSNAIDEGDGCFNLLHKCNDSC METIRNGTYNHEDYR EESQLKRQEIEGIKLK TE [SEQ ID NO.: 97]

In certain embodiments, the influenza hemagglutinin stem domainpolypeptides comprise one or more immunogenic epitopes in the tertiaryor quaternary structure of an influenza hemagglutinin polypeptide.

In certain embodiments, the HA1 N-terminal stem segment comprises theamino acid sequence A₁₇-A₁₈-(Xaa)_(n)-A₃₈ (SEQ ID NO:146), wherein

A₁₇ is Y or H;

A₁₈ is H, L, or Q;

(Xaa)_(n) represents a sequence of 18-20 amino acid residues; and

A₃₈ is H, S, Q, T or N.

In certain embodiments, the HA1 C-terminal stem segment comprises theamino acid sequence A₂₉₁-A₂₉₂ (SEQ ID NO:147), wherein

A₂₉₁ is T, S, N, D, P or K; and

A₂₉₂ is L, M, K or R.

In certain embodiments, the HA2 domain comprises the amino acid sequenceA₁₈-A₁₉-A₂₀-A₂₁(SEQ ID NO:148), wherein

A₁₈ is V or I;

A₁₉ is D, N or A;

A₂₀ is G, and

A₂₁ is W.

In certain embodiments, the HA2 domain comprises the amino acid sequenceA₃₈-A₃₉-A₄₀-A₄₁-A₄₂-A₄₃-A₄₄-A₄₅-A₄₆-A₄₇-A₄₈-A₄₉-A₅₀-A₅₁-A₅₂-A₅₃-A₅₄-A₅₅-A₅₆(SEQID NO:149), wherein

A₃₈ is K, Q, R, L or Y;

A₃₉ is any amino acid residue;

A₄₀ is any amino acid residue;

A₄₁ is T;

A₄₂ is Q;

A₄₃ is any amino acid residue;

A₄₄ is A;

A₄₅ is I;

A₄₆ is D;

A₄₇ is any amino acid residue;

A₄₈ is I, V or M;

A₄₉ is T, Q or N;

A₅₀ is any amino acid residue;

A₅₁ is K;

A₅₂ is V or L;

A₅₃ is N;

A₅₄ is any amino acid residue;

A₅₅ is V, I or L; and

A₅₆ is V or I.

In certain embodiments, the influenza stem domain polypeptides comprisetwo amino acid sequences selected from SEQ ID NOS:146-149. In certainembodiments, the influenza stem domain polypeptides comprise three aminoacid sequences selected from SEQ ID NOS:146-149. In certain embodiments,the influenza stem domain polypeptides comprise four amino acidsequences selected from SEQ ID NOS:146-149.

In certain embodiments, the HA1 N-terminal stem segments are based on aninfluenza B hemagglutinin. In certain embodiments, the HA1 N-terminalstem segment is selected from SEQ ID NOS:154-157, presented in Table 3below.

In certain embodiments, the HA1 C-terminal stem segments are based on aninfluenza B hemagglutinin. In certain embodiments, the HA1 C-terminalstem segment is selected from SEQ ID NOS:158-159, presented in Table 3below.

In certain embodiments, the HA2 stem domains are based on an influenza Bhemagglutinin. Exemplary residues for the end of an N-terminal stemsegment and the end of a C-terminal stem segment of an influenza Bhemagglutinin are indicated in FIG. 2. In certain embodiments, the HA2stem domain is according to SEQ ID NO:160, presented in Tables 3 and 4below.

In particular embodiments, the boundaries of the influenza B virus HA1N-terminal stem segment and influenza B virus HA1 C-terminal segment aredefined with respect to three pairs of amino acid residues: Arg₅₀ andSer₂₇₇; Ala₆₆ and Trp₂₇₁; and Lys₈₀ and Ser₂₇₇. The residue numbers arebased on the numbering of the B-HA from influenza virus B as describedin Protein Data Bank accession No. 3BT6. The amino acid sequencecorresponding to the X-ray crystal structure of the B-HA protein inProtein Data Bank accession No. 3BT6 is aligned with representative H1and H3 amino acid sequence and shown in FIG. 2. Positions of the threepairs of residues are also highlighted in FIG. 2.

In certain embodiments, an influenza B virus HA1 N-terminal stem segmentstarts at residue 1 (based on numbering of an influenza B virus HA1subunit as in PDB file 3BT6) and ends at Arg₅₀. In certain embodiments,an influenza B virus HA1 N-terminal stem segment starts at residue 1 andends at Ala₆₆. In some embodiments, an influenza B virus HA1 N-terminalstem segment starts at residue 1 and ends at Lys₈₀. In some embodiments,an influenza B virus N-terminal stem segment starts at residue 1 andends at Arg₈₀.

In some embodiments, an influenza B virus HA1 N-terminal stem segmenthas an amino acid sequence according to any one of SEQ ID NOS:154-157,as illustrated in TABLE 3. In some embodiments, an influenza B virus HA1N-terminal stem segment has an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96% or 98% identical to any one of the aminoacid sequences of any one of SEQ ID NOS:154-157.

In some embodiments, an influenza B virus HA1 N-terminal stem segmenthas an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%,95%, 96% or 98% identical to the amino acid sequence SEQ ID NO:154,which corresponds to residues 1-50 of the influenza B virus HA1.

In some embodiments, an influenza B virus HA1 N-terminal stem segmenthas an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%,95%, 96% or 98% identical to the amino acid sequence SEQ ID NO:155,which corresponds to residues 1-66 of the influenza B virus HA1.

In some embodiments, an influenza B virus HA1 N-terminal stem segmenthas an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%,95%, 96% or 98% identical to the amino acid sequence SEQ ID NO:156,which corresponds to residues 1-80 of the influenza B virus HA1.

In some embodiments, an influenza B virus HA1 N-terminal stem segmenthas an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%,95%, 96% or 98% identical to the amino acid sequence SEQ ID NO:157,which corresponds to residues 1-80 of the influenza B virus HA1.

In some embodiments, an influenza B virus HA1 C-terminal stem segmenthas an amino acid sequence that starts at Ser₂₇₇ residue or Trp₂₇₁, orcorresponding residues in other influenza B virus HA subtypes.

In some embodiments, an influenza B virus HA1 C-terminal stem segmenthas an amino acid sequence according to any one of SEQ ID NOS:158-159,as illustrated in TABLE 3. In some embodiments, an influenza B virus HA1C-terminal stem segment has an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96% or 98% identical to SEQ ID NO:158, whichcorrespond to residues 277-344 of influenza B virus HA1. In someembodiments, an influenza B virus HA1 C-terminal stem segment has anamino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%or 98% identical to SEQ ID NO.:159, which correspond to residues 271-344of influenza B virus HA1.

In some embodiments, an influenza B virus HA1 C-terminal stem segmentstarts at residue-276, residue-275, residue-274, residue-273, orresidue-272. In other embodiments, an influenza B virus HA1 C-terminalstem segment starts at residue-278, residue-279, residue-280,residue-281, or residue-282.

In certain embodiments, the influenza B virus HA2 domain is in tertiaryor quaternary association with the influenza B virus HA1 domain throughthe influenza B virus HA1 N-terminal segment, the influenza B virus HA1C-terminal segment, or both.

In some embodiments, the influenza B virus HA1 C-terminal segment andthe influenza B virus HA2 subunit are covalently linked. For example, atits C-terminus (e.g., at the ending residue of the second sequence), theinfluenza B virus HA1 C-terminal segment is covalently linked to theinfluenza B virus HA2 domain in such embodiments. In some embodiments,the influenza B virus HA1 C-terminal segment and influenza B virus HA2domain form a continuous polypeptide chain.

In some embodiments, the influenza B virus HA2 domain has the amino acidsequence of SEQ ID NO:160 or 161, as illustrated in TABLE 3 or 4. Insome embodiments, the amino acid sequence of the HA2 domain is at least70%, 75%, 80%, 85%, 90%, 95%, 96% or 98% identical to any one of SEQ IDNOS:160-161.

In certain embodiments, the influenza B stem domain polypeptidescomprise a signal peptide. The signal peptide can be any signal peptidedeemed suitable to those of skill in the art, including any signalpeptide described herein. In certain embodiments, the signal peptide isat least 70%, 75%, 80%, 85%, 90%, 95%, 96% or 98% identical to any ofSEQ ID NOS:150-153. In certain embodiments, the signal peptide isaccording to any of SEQ ID NOS:150-153.

In certain embodiments, the influenza B stem domain polypeptidescomprise a luminal domain. The luminal domain can be any luminal domaindeemed suitable to those of skill in the art, including any luminaldomain described herein. In certain embodiments, the luminal is at least60% or 80%, identical to SEQ ID NO:162. In certain embodiments, theluminal domain is according to SEQ ID NO:162.

In certain embodiments, the influenza B stem domain polypeptidescomprise a transmembrane domain. The transmembrane domain can be anytransmembrane domain deemed suitable to those of skill in the art,including any transmembrane domain described herein. In certainembodiments, the transmembrane domain is at least 70%, 75%, 80%, 85%,90%, 95%, 96% or 98% identical to SEQ ID NO:163. In certain embodiments,the transmembrane domain is according to SEQ ID NO:163.

In certain embodiments, the influenza B stem domain polypeptidescomprise a cytoplasmic domain. The cytoplasmic domain can be anycytoplasmic domain deemed suitable to those of skill in the art,including any cytoplasmic domain described herein. In certainembodiments, the cytoplasmic domain is at least 70%, 75%, 80%, 85%, 90%,95%, 96% or 98% identical to SEQ ID NO:164. In certain embodiments, thecytoplasmic domain is according to SEQ ID NO:164.

TABLE 3 Exemplary Influenza B Hemagglutinin Sequences HA HA1 HA1construct Signal N-terminal C-terminal variants peptide Stem SegmentStem Segment HA2 Domain Arg50- MKAIIVILMV DRICTGITSSNS SKVIKGSLPLIGFFGAIAGFLEGG Ser277 VTSNA PHVVKTATQG GEADCLHEKY WEGMIAGWHGY [SEQ IDEVNVTGVIPLT GGLNKSKPYY TSHGAHGVAVAA NO.: 150] TTPTKSHFANL TGEHAKAIGNDLKSTQEAINKIT KGTETR CPIWVKTPLKL KNLNSLSELEVKN [SEQ ID ANGTKYRPPALQRLSGAMDELH NO.: 154] KLLKER NEILELDEKVDDL [SEQ ID RADTISSQIELAVLNO.: 158] LSNEGIINSEDEHL LALERKLKKMLG PSAVEIGNGCFET KHKCNQTCLDRIAAGTFDAGEFSLP TFDSLNITAASLN DDGLDNHTILLYY STAASSLAVTLMI AIFVVYMVSRDNVSCSICL [SEQ ID NO.: 160] Ala66- MKAIIVILMV DRICTGITSSNS WCASGRSKVIGFFGAIAGFLEGG Trp271 VTSNA PHVVKTATQG KGSLPLIGEAD WEGMIAGWHGY [SEQ IDEVNVTGVIPLT CLHEKYGGLN TSHGAHGVAVAA NO.: 151] TTPTKSHFANL KSKPYYTGEHDLKSTQEAINKIT KGTETRGKLC AKAIGNCPIW KNLNSLSELEVKN PKCLNCTDLD VKTPLKLANGLQRLSGAMDELH VA TKYRPPAKLL NEILELDEKVDDL [SEQ ID KER RADTISSQIELAVLNO.: 155] [SEQ ID LSNEGIINSEDEHL NO.: 159] LALERKLKKMLG PSAVEIGNGCFETKHKCNQTCLDRI AAGTFDAGEFSLP TFDSLNITAASLN DDGLDNHTILLYY STAASSLAVTLMIAIFVVYMVSRDN VSCSICL [SEQ ID NO.: 160] Lys80- MKAIIVILMV DRICTGITSSNSSKVIKGSLPLI GFFGAIAGFLEGG Ser277 VTSNA PHVVKTATQG GEADCLHEKY WEGMIAGWHGY[SEQ ID EVNVTGVIPLT GGLNKSKPYY TSHGAHGVAVAA NO.: 152] TTPTKSHFANLTGEHAKAIGN DLKSTQEAINKIT KGTETRGKLC CPIWVKTPLKL KNLNSLSELEVKN PKCLNCTDLDANGTKYRPPA LQRLSGAMDELH VALGRPKCTG KLLKER NEILELDEKVDDL KIPSAK [SEQ IDRADTISSQIELAVL [SEQ ID NO.: 158] LSNEGIINSEDEHL NO.: 156] LALERKLKKMLGPSAVEIGNGCFET KHKCNQTCLDRI AAGTFDAGEFSLP TFDSLNITAASLN DDGLDNHTILLYYSTAASSLAVTLMI AIFVVYMVSRDN VSCSICL [SEQ ID NO.: 160] Arg80- MKAIIVILMVDRICTGITSSNS SKVIKGSLPLI GFFGAIAGFLEGG Ser277 VTSNA PHVVKTATQGGEADCLHEKY WEGMIAGWHGY [SEQ ID EVNVTGVIPLT GGLNKSKPYY TSHGAHGVAVAANO.: 153] TTPTKSHFANL TGEHAKAIGN DLKSTQEAINKIT KGTETRGKLC CPIWVKTPLKLKNLNSLSELEVKN PKCLNCTDLD ANGTKYRPPA LQRLSGAMDELH VALGRPKCTG KLLKERNEILELDEKVDDL KIPSAR [SEQ ID RADTISSQIELAVL [SEQ ID NO.: 158]LSNEGIINSEDEHL NO.: 157] LALERKLKKMLG PSAVEIGNGCFET KHKCNQTCLDRIAAGTFDAGEFSLP TFDSLNITAASLN DDGLDNHTILLYY STAASSLAVTLMI AIFVVYMVSRDNVSCSICL [SEQ ID NO.: 160]

Table 4 provides the putative stem domain, luminal domain, transmembranedomain and cytoplasmic domain of HA from influenza B.

TABLE 4 Exemplary Influenza B Hemagglutinin Sequences HA2 domain Subtype(Genbank Luminal Transmembrane Cytoplasmic No.) Stem domain domaindomain domain HA2 GFFGAIAGFLEG DGLDN HTILLYYSTAAS SRDNVSCSIC (AY096185)GWEGMIAGWH [SEQ ID SLAVTLMIAIFV L GYTSHGAHGV NO.: 162] VYMV [SEQ IDAVAADLKSTQE [SEQ ID NO.: 164] AINKITKNLNSL NO.: 163] SELEVKNLQRLSGAMDELHNEI LELDEKVDDLR ADTISSQIELAV LLSNEGIINSED EHLLALERKLKKMLGPSAVEIG NGCFETKHKCN QTCLDRIAAGT FDAGEFSLPTFD SLNITAASLND [SEQ IDNO.: 161]

As illustrated in FIGS. 1 and 2, HA1 N-terminal stem segments sharesequence identity between influenza A and influenza B and additionallyacross influenza A subtypes. Similarly, HA1 C-terminal stem segmentsalso share sequence identity between influenza A and influenza B andadditionally across influenza A subtypes. Further, HA2 domains alsoshare sequence identity between influenza A and influenza B andadditionally across influenza A subtypes.

In some embodiments, the influenza hemagglutinin stem domain polypeptideis a hybrid polypeptide that comprises or consists essentially ofsegments and/or domains from a plurality of influenza strains orsubtypes. For example, an influenza hemagglutinin stem domainpolypeptide might comprise HA1 N-terminal and HA1 C-terminal stemsegments from different influenza A virus HA subtypes. In someembodiments, the HA1 N-terminal stem segment is from influenza A viruswhile the HA1 C-terminal stem segment is from influenza B virus.Similarly, HA2 may also be from influenza A virus while the HA1N-terminal and/or C-terminal stem segment is from influenza B virus.

It will be understood that any combination of the sequence elementslisted in Tables 1-4 or the variants thereof may be used to form thehemagglutinin HA stem domain polypeptides of the present invention.

In an influenza stem domain polypeptide provided herein, a linkercovalently connects the HA1 N-terminal stem segment to the HA1C-terminal stem segment. In certain embodiments, the linker is a directbond. In certain embodiments, the linker is a peptide that comprises oneamino acid residue, two or fewer amino acid residues, three or feweramino acid residues, four or fewer amino acid residues, five or feweramino acid residues, ten or fewer amino acid residues, 15 or fewer aminoacid residues, 20 or fewer amino acid residues, 30 or fewer amino acidresidues, 40 or fewer amino acid residues, or 50 or fewer amino acidresidues. In certain embodiments, the linker peptide comprises 50 ormore amino acid residues. In certain embodiments the linkersubstantially lacks a globular head domain. In other words, the linkercomprises no more than 10, 9, 8, 7, 6, 5 or 4 contiguous, sequentialamino acid residues from the amino acid sequence of an influenzaglobular head domain. In certain embodiments, the linker is other thanLys-Leu-Asn-Gly-Ser-Gly-Ile-Met-Lys-Thr-Glu-Gly-Thr-Leu-Glu-Asn (SEQ IDNO:311). In certain embodiments, the linker is other thanAsn-Asn-Ile-Asp-Thr orLys-Leu-Asn-Gly-Ser-Gly-Ile-Met-Lys-Thr-Glu-Gly-Thr-Leu-Glu-Asn (SEQ IDNO:312). In certain embodiments, the linker is other thanAsn-Asn-Ile-Asp-Thr (SEQ ID NO:315).

In certain embodiments, the linker is covalently connected, at one end,to the C-terminus of the HA1 N-terminal stem segment. The linker peptideis also covalently connected, at the other end, to the N-terminus of theHA1 C-terminal stem segment. In certain embodiments, one of the covalentlinks is an amide bond. In certain embodiments, both covalent links areamide bonds.

The linker might be any linker deemed suitable by one of skill in theart. In certain embodiments, the linker is selected based on the HA1N-terminal stem segment and the HA1 C-terminal stem segment. In theseembodiments, the linker might be selected with molecular modelingprograms such as InsightII and Quanta, both from Accelrys. In certainembodiments, the linker is a structural motif that allows structuralalignment of the HA1 N-terminal stem segment and the HA1 C-terminal stemsegment that is consistent with the structure of a hemagglutinin stemdomain as recognized by those of skill in the art. In certainembodiments, the linker is selected from a library of candidate linkers.In certain embodiments, the library includes three dimensionalpolypeptide structures in a publicly available database such as theProtein Data Bank (PDB) or the Macromolecular Structure Database at theEuropean Molecular Biology Laboratory (EMBL) or European BioinformaticsInstitute (EBI). In certain embodiments, the library includesproprietary three-dimensional polypeptide structures associated withcommercial programs such as InsightII and Quanta, both from Accelrys.Additionally, any databases or collections of protein structures orstructural elements can be used to select the linker. Exemplary databaseor collections of protein structural elements include but are notlimited to the Structural Classification of Proteins (SCOP, maintainedby and available through Cambridge University); the database of proteinfamilies (Pfam, maintained by and available through the Wellcome TrustSanger Institute); the Universal Protein Resource (UniProt, maintainedby and available through the UniProt Consortium); the Integratedresource for protein families (InterPro; maintained by and availablethrough EMBL-EBI); the Class Architecture Topology Homologoussuperfamily (CATH, maintained by and available through Institute ofStructural and Molecular Biology at the University College London); andthe families of structurally similar proteins (FSSP, maintained by andavailable through EBI). Any algorithm deemed suitable by one of skill inthe art may be used to select the linker, including but not limited bythose used by SCOP, CATH and FSSP. Useful examples include but are notlimited to Pymol (Delano Scientific LLC), InsightII and Quanta (bothfrom Accelrys), MIDAS (University of California, San Francisco),SwissPDB viewer (Swiss Institute of Bioinformatics), TOPOFIT(Northeastern University), CBSU LOOPP (Cornell University), andSuperPose (University of Alberta, Edmonton).

In certain embodiments, the linker is a direct bond. In certainembodiments, the linker is selected from the group consisting of Gly,Gly-Gly, Gly-Gly-Gly, Gly-Gly-Gly-Gly (SEQ ID NO:319) andGly-Gly-Gly-Gly-Gly (SEQ ID NO:320). In certain embodiments, the linkeris selected from the group consisting of Gly-Pro and Pro-Gly. In certainembodiments, the linker is a 281 turn loop, e.g. having the sequenceITPNGSIPNDKPFQNVNKITYGA (SEQ ID NO:165).

In certain embodiments the linker comprises a glycosylation sequence. Incertain embodiments, the linker comprises an amino acid sequenceaccording to Asn-Xaa-Ser/Thr where Xaa is any amino acid other thanproline and Ser/Thr is serine or threonine. In certain embodiments, thelinker comprises the amino acid sequence Asn-Ala-Ser. In certainembodiments the linker is a glycosylation sequence. In certainembodiments, the linker is an amino acid sequence according toAsn-Xaa-Ser/Thr where Xaa is any amino acid other than proline andSer/Thr is serine or threonine. In certain embodiments, the linker isthe amino acid sequence Asn-Ala-Ser.

In certain embodiments, influenza hemagglutinin stem domain polypeptidesare capable of forming a three dimensional structure that is similar tothe three dimensional structure of the stem domain of a native influenzahemagglutinin. Structural similarity might be evaluated based on anytechnique deemed suitable by those of skill in the art. For instance,reaction, e.g. under non-denaturing conditions, of an influenzahemagglutinin stem domain polypeptide with a neutralizing antibody orantiserum that recognizes a native influenza hemagglutinin mightindicate structural similarity. Useful neutralizing antibodies orantisera are described in, e.g., Sui, et al., 2009, Nat. Struct. Mol.Biol. 16(3):265-273, Ekiert et al., Feb. 26, 2009, Science [DOI:10.1126/science.1171491], Wang et al. (2010) “Broadly ProtectiveMonoclonal Antibodies against H3 Influenza Viruses following SequentialImmunization with Different Hemagglutinins,” PLOS Pathogens 6(2):1-9,and Kashyap et al., 2008, Proc. Natl. Acad. Sci. USA 105(16):5986-5991,the contents of which are hereby incorporated by reference in theirentireties. In certain embodiments, the antibody or antiserum is anantibody or antiserum that reacts with a non-contiguous epitope (i.e.,not contiguous in primary sequence) that is formed by the tertiary orquaternary structure of a hemagglutinin.

In certain embodiments, structural similarity might be assessed byspectroscopic techniques such as circular dichroism, Raman spectroscopy,NMR, 3D NMR and X-ray crystallography. Known influenza hemagglutininstructures determined by X-ray crystallography are described instructural coordinates in Protein Data Bank files including but notlimited to 1HGJ (an HA H3N2 strain) and 1RUZ (an HA H1N1 strain).

In certain embodiments, structural similarity is evaluated by RMSdeviation between corresponding superimposed portions of two structures.In order to create a meaningful superimposition, in certain embodimentsthe coordinates of at least 20 corresponding atoms, 25 correspondingatoms, 30 corresponding atoms, 40 corresponding atoms, 50 correspondingatoms, 60 corresponding atoms, 70 corresponding atoms, 80 correspondingatoms, 90 corresponding atoms, 100 corresponding atoms, 120corresponding atoms, 150 corresponding atoms, 200 corresponding atoms,or 250 corresponding atoms are used to calculate an RMS deviation.

In certain embodiments, the coordinates of all corresponding atoms inamino acid backbones are used to calculate an RMS deviation. In certainembodiments, the coordinates of all corresponding alpha carbon-atoms inthe amino acid backbones are used to calculate an RMS deviation. Incertain embodiments, the coordinates of all corresponding identicalresidues, including side chains, are used to calculate an RMS deviation.

In certain embodiments, coordinates of all or a portion of thecorresponding atoms in a HA1 N-terminal segment are used to calculate anRMS deviation. In certain embodiments, coordinates of all or a portionof the corresponding atoms in a HA1 C-terminal segment are used tocalculate an RMS deviation. In certain embodiments, coordinates of allor a portion of the corresponding atoms in both a HA1 N-terminal segmentand a C-terminal segment are used to calculate an RMS deviation. Incertain embodiments, coordinates of all or a portion of correspondingatoms in HA2 domains are used to calculate an RMS deviation.

In certain embodiments, the RMS deviation between the structures of ainfluenza hemagglutinin stem domain polypeptide and correspondingportions of a known influenza A virus hemagglutinin stem domain (e.g.,from 1HGJ or 1RUZ) is 5 Å or less, 4 Å or less, 3 Å or less, 2.5 Å orless, 2 Å or less, 1.5 Å or less, 1 Å or less, 0.75 Å or less, 0.5 Å orless, 0.3 Å or less, 0.2 Å or less, or 0.1 Å or less. Commerciallyavailable or open source software might be used to perform thestructural superimpositions and/or RMS deviation calculations. Usefulexamples include but are not limited to Pymol (Delano Scientific LLC),InsightII and Quanta (both from Accelrys), MIDAS (University ofCalifornia, San Francisco), SwissPDB viewer (Swiss Institute ofBioinformatics), TOPOFIT (Northeastern University), CBSU LOOPP (CornellUniversity), and SuperPose (University of Alberta, Edmonton).

In certain embodiments, any influenza hemagglutinin stem domainpolypeptide provided herein can further comprise one or more polypeptidedomains deemed suitable to those of skill in the art. Useful polypeptidedomains include domains that facilitate purification, folding andcleavage of portions of a polypeptide. For example, a His tag(His-His-His-His-His-His, SEQ ID NO:166), FLAG epitope or otherpurification tag can facilitate purification of a polypeptide providedherein. A foldon, or trimerization, domain from bacteriophage T4fibritin can facilitate trimerization of polypeptides provided herein.The foldon domain can have any foldon sequence known to those of skillin the art (see, e.g., Papanikolopoulou et al., 2004, J. Biol. Chem.279(10):8991-8998, the contents of which are hereby incorporated byreference in their entirety. Examples includeGSGYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO:167). A foldon domain can beuseful to facilitate trimerization of soluble polypeptides providedherein. Cleavage sites can be used to facilitate cleavage of a portionof a polypeptide, for example cleavage of a purification tag or foldondomain or both. Useful cleavage sites include a thrombin cleavage site,for example one with the sequence LVPRGSP (SEQ ID NO:168).

In certain embodiments, provided are influenza hemagglutinin stem domainpolypeptides comprising an elastase cleavage site. Those of skill in theart will recognize that the trypsin cleavage site at the linkage betweenHA1 and HA2 can be mutated to an elastase cleavage site by substitutingvaline for the arginine or lysine at the HA1-HA2 cleavage site in ahemagglutinin sequence (see, e.g., Stech et al., 2005, Nature Med.11(6):683-689). Accordingly, provided herein are influenza hemagglutininstem domain polypeptides having a valine substitution at the C-terminusof the C-terminal stem segment (i.e., the C-terminus of the HA1 domain).In particular embodiments, provided herein are influenza hemagglutininstem domain polypeptides comprising any of SEQ ID NOS:50-65 or 158-159wherein the C-terminal amino acid residue, e.g. arginine or lysine, ofSEQ ID NOS:50-65 or 158-159 is substituted with a valine residue.

In certain embodiments, provided herein are influenza hemagglutinin stemdomain polypeptides that are predicted to be resistant to proteasecleavage at the junction between HA1 and HA2. Those of skill in the artshould recognize that the Arg-Gly sequence spanning HA1 and HA2 is arecognition site for trypsin and is typically cleaved for hemagglutininactivation. Since the stem domain polypeptides described herein need notbe activated, provided herein are influenza hemagglutinin stem domainpolypeptides that are predicted to be resistant to protease cleavage. Incertain embodiments, provided is any influenza hemagglutinin stem domainpolypeptide described herein wherein the protease site spanning HA1 andHA2 is mutated to a sequence that is resistant to protease cleavage. Incertain embodiments, provided is any influenza hemagglutinin stem domainpolypeptide described herein wherein the C-terminal residue of the HA1C-terminal stem segment is any residue other than Lys or Arg. In certainembodiments, provided is any influenza hemagglutinin stem domainpolypeptide described herein wherein the N-terminal residue of the HA2domain is proline. In certain embodiments, provided is any influenzahemagglutinin stem domain polypeptide described herein wherein theC-terminal residue of the HA1 C-terminal stem segment is Ala and theN-terminal residue of the HA2 domain is also Ala.

In certain embodiments, provided herein are influenza hemagglutinin stemdomain polypeptides consisting of an HA1 N-terminal stem segmentcovalently linked to a linker, in turn covalently linked to an HA1C-terminal stem segment in binding association with an HA2 stem domain.In certain embodiments, provided herein are influenza hemagglutinin stemdomain polypeptides consisting of an HA1 N-terminal stem segmentcovalently linked to a linker, in turn covalently linked to an HA1C-terminal stem segment, in turn covalently linked to an HA2 stemdomain. In certain embodiments, provided herein are influenzahemagglutinin stem domain polypeptides consisting of a signal peptidecovalently linked to an HA1 N-terminal stem segment covalently linked toa linker, in turn covalently linked to an HA1 C-terminal stem segment,in turn covalently linked to an HA2 stem domain.

In certain embodiments, provided herein are influenza hemagglutinin stemdomain polypeptides consisting of an HA1 N-terminal stem segmentcovalently linked to a linker, in turn covalently linked to an HA1C-terminal stem segment in binding association with an HA2 stem domainthat is covalently linked to an HA2 luminal domain. In certainembodiments, provided herein are influenza hemagglutinin stem domainpolypeptides consisting of an HA1 N-terminal stem segment covalentlylinked to a linker, in turn covalently linked to an HA1 C-terminal stemsegment, in turn covalently linked to an HA2 stem domain that iscovalently linked to an HA2 luminal domain. In certain embodiments,provided herein are influenza hemagglutinin stem domain polypeptidesconsisting of a signal peptide covalently linked to an HA1 N-terminalstem segment covalently linked to a linker, in turn covalently linked toan HA1 C-terminal stem segment, in turn covalently linked to an HA2 stemdomain that is covalently linked to an HA2 luminal domain.

In certain embodiments, provided herein are influenza hemagglutinin stemdomain polypeptides consisting of an HA1 N-terminal stem segmentcovalently linked to a linker, in turn covalently linked to an HA1C-terminal stem segment in binding association with an HA2 stem domainthat is covalently linked to, in sequence, a thrombin cleavage site, afoldon domain and a His tag. In certain embodiments, provided herein areinfluenza hemagglutinin stem domain polypeptides consisting of an HA1N-terminal stem segment covalently linked to a linker, in turncovalently linked to an HA1 C-terminal stem segment, in turn covalentlylinked to an HA2 stem domain that is covalently linked to, in sequence,a thrombin cleavage site, a foldon domain and a His tag. In certainembodiments, provided herein are influenza hemagglutinin stem domainpolypeptides consisting of a signal peptide covalently linked to an HA1N-terminal stem segment covalently linked to a linker, in turncovalently linked to an HA1 C-terminal stem segment, in turn covalentlylinked to an HA2 stem domain that is covalently linked to, in sequence,a thrombin cleavage site, a foldon domain and a His tag.

In certain embodiments, provided herein are influenza hemagglutinin stemdomain polypeptides consisting of an HA1 N-terminal stem segmentcovalently linked to a linker, in turn covalently linked to an HA1C-terminal stem segment in binding association with an HA2 stem domainthat is covalently linked to an HA2 luminal domain that is covalentlylinked to, in sequence, a thrombin cleavage site, a foldon domain and aHis tag. In certain embodiments, provided herein are influenzahemagglutinin stem domain polypeptides consisting of an HA1 N-terminalstem segment covalently linked to a linker, in turn covalently linked toan HA1 C-terminal stem segment, in turn covalently linked to an HA2 stemdomain that is covalently linked to an HA2 luminal domain that iscovalently linked to, in sequence, a thrombin cleavage site, a foldondomain and a His tag. In certain embodiments, provided herein areinfluenza hemagglutinin stem domain polypeptides consisting of a signalpeptide covalently linked to an HA1 N-terminal stem segment covalentlylinked to a linker, in turn covalently linked to an HA1 C-terminal stemsegment, in turn covalently linked to an HA2 stem domain that iscovalently linked to an HA2 luminal domain that is covalently linked to,in sequence, a thrombin cleavage site, a foldon domain and a His tag.

In certain embodiments, provided herein are influenza hemagglutinin stemdomain polypeptides consisting of an HA1 N-terminal stem segmentcovalently linked to a linker, in turn covalently linked to an HA1C-terminal stem segment in binding association with an HA2 stem domainthat is covalently linked to an HA2 luminal domain that is in turncovalently linked to an HA2 transmembrane domain. In certainembodiments, provided herein are influenza hemagglutinin stem domainpolypeptides consisting of an HA1 N-terminal stem segment covalentlylinked to a linker, in turn covalently linked to an HA1 C-terminal stemsegment, in turn covalently linked to an HA2 stem domain that iscovalently linked to an HA2 luminal domain that is in turn covalentlylinked to an HA2 transmembrane domain. In certain embodiments, providedherein are influenza hemagglutinin stem domain polypeptides consistingof a signal peptide covalently linked to an HA1 N-terminal stem segmentcovalently linked to a linker, in turn covalently linked to an HA1C-terminal stem segment, in turn covalently linked to an HA2 stem domainthat is covalently linked to an HA2 luminal domain that is in turncovalently linked to an HA2 transmembrane domain.

In certain embodiments, provided herein are influenza hemagglutinin stemdomain polypeptides consisting of an HA1 N-terminal stem segmentcovalently linked to a linker, in turn covalently linked to an HA1C-terminal stem segment in binding association with an HA2 stem domainthat is covalently linked to an HA2 luminal domain that is in turncovalently linked to an HA2 transmembrane domain that is in turncovalently linked to an HA2 cytoplasmic domain. In certain embodiments,provided herein are influenza hemagglutinin stem domain polypeptidesconsisting of an HA1 N-terminal stem segment covalently linked to alinker, in turn covalently linked to an HA1 C-terminal stem segment, inturn covalently linked to an HA2 stem domain that is covalently linkedto an HA2 luminal domain that is in turn covalently linked to an HA2transmembrane domain that is in turn covalently linked to an HA2cytoplasmic domain. In certain embodiments, provided herein areinfluenza hemagglutinin stem domain polypeptides consisting of a signalpeptide covalently linked to an HA1 N-terminal stem segment covalentlylinked to a linker, in turn covalently linked to an HA1 C-terminal stemsegment, in turn covalently linked to an HA2 stem domain that iscovalently linked to an HA2 luminal domain that is in turn covalentlylinked to an HA2 transmembrane domain that is in turn covalently linkedto an HA2 cytoplasmic domain.

In certain embodiments, provided herein is an influenza hemagglutininstem domain polypeptide having a sequence selected from the groupconsisting of:

(SEQ ID NO:34)-LL-(SEQ ID NO:50)-(SEQ ID NO:66),

(SEQ ID NO:35)-LL-(SEQ ID NO:51)-(SEQ ID NO:67),

(SEQ ID NO:36)-LL-(SEQ ID NO:52)-(SEQ ID NO:68),

(SEQ ID NO:37)-LL-(SEQ ID NO:53)-(SEQ ID NO:69),

(SEQ ID NO:38)-LL-(SEQ ID NO:54)-(SEQ ID NO:70),

(SEQ ID NO:39)-LL-(SEQ ID NO:55)-(SEQ ID NO:71),

(SEQ ID NO:40)-LL-(SEQ ID NO:56)-(SEQ ID NO:72),

(SEQ ID NO:41)-LL-(SEQ ID NO:57)-(SEQ ID NO:73),

(SEQ ID NO:42)-LL-(SEQ ID NO:58)-(SEQ ID NO:74),

(SEQ ID NO:43)-LL-(SEQ ID NO:59)-(SEQ ID NO:75),

(SEQ ID NO:44)-LL-(SEQ ID NO:60)-(SEQ ID NO:76),

(SEQ ID NO:45)-LL-(SEQ ID NO:61)-(SEQ ID NO:77),

(SEQ ID NO:46)-LL-(SEQ ID NO:62)-(SEQ ID NO:78),

(SEQ ID NO:47)-LL-(SEQ ID NO:63)-(SEQ ID NO:79),

(SEQ ID NO:48)-LL-(SEQ ID NO:64)-(SEQ ID NO:80), and

(SEQ ID NO:49)-LL-(SEQ ID NO:65)-(SEQ ID NO:81),

wherein each sequence above is linked to the adjacent sequence asdescribed herein and wherein LL is a linker as described herein. Inparticular, the HA1 C-terminal segments can be covalently ornon-covalently linked to the HA2 domains. In certain embodiments, LL isselected from the group consisting of a direct bond, Gly, Gly-Gly,Gly-Gly-Gly, Gly-Gly-Gly-Gly (SEQ ID NO:319), (Gly)n (wherein n is anynumber of Glycine residues so long as there is flexibility in thepeptide linker; in certain embodiments, n is 2, 3, 4, 5, 6, or 7 Glycineresidues), Gly-Pro, ITPNGSIPNDKPFQNVNKITYGA (SEQ ID NO:165) andAsn-Ala-Ser.

In certain embodiments, provided herein is an influenza hemagglutininstem domain polypeptide having a sequence selected from the groupconsisting of:

(SEQ ID NO:34)-LL-(SEQ ID NO:50)-(SEQ ID NO:82),

(SEQ ID NO:35)-LL-(SEQ ID NO:51)-(SEQ ID NO:83),

(SEQ ID NO:36)-LL-(SEQ ID NO:52)-(SEQ ID NO:84),

(SEQ ID NO:37)-LL-(SEQ ID NO:53)-(SEQ ID NO:85),

(SEQ ID NO:38)-LL-(SEQ ID NO:54)-(SEQ ID NO:86),

(SEQ ID NO:39)-LL-(SEQ ID NO:55)-(SEQ ID NO:87),

(SEQ ID NO:40)-LL-(SEQ ID NO:56)-(SEQ ID NO:88),

(SEQ ID NO:41)-LL-(SEQ ID NO:57)-(SEQ ID NO:89),

(SEQ ID NO:42)-LL-(SEQ ID NO:58)-(SEQ ID NO:90),

(SEQ ID NO:43)-LL-(SEQ ID NO:59)-(SEQ ID NO:91),

(SEQ ID NO:44)-LL-(SEQ ID NO:60)-(SEQ ID NO:92),

(SEQ ID NO:45)-LL-(SEQ ID NO:61)-(SEQ ID NO:93),

(SEQ ID NO:46)-LL-(SEQ ID NO:62)-(SEQ ID NO:94),

(SEQ ID NO:47)-LL-(SEQ ID NO:63)-(SEQ ID NO:95),

(SEQ ID NO:48)-LL-(SEQ ID NO:64)-(SEQ ID NO:96), and

(SEQ ID NO:49)-LL-(SEQ ID NO:65)-(SEQ ID NO:97),

wherein each sequence above is linked to the adjacent sequence asdescribed herein and wherein LL is a linker as described herein. Inparticular, the HA1 C-terminal segments can be covalently ornon-covalently linked to the HA2 domains. In certain embodiments, LL isselected from the group consisting of a direct bond, Gly, Gly-Gly,Gly-Gly-Gly, Gly-Gly-Gly-Gly (SEQ ID NO:319), (Gly)n, Gly-Pro,ITPNGSIPNDKPFQNVNKITYGA (SEQ ID NO:165) and Asn-Ala-Ser.

In certain embodiments, provided herein is an influenza hemagglutininstem domain polypeptide having a sequence selected from the groupconsisting of:

(SEQ ID NO:34)-LL-(SEQ ID NO:50)-(SEQ ID NO:82)-(SEQ ID NO:98),

(SEQ ID NO:35)-LL-(SEQ ID NO:51)-(SEQ ID NO:83)-(SEQ ID NO:99),

(SEQ ID NO:36)-LL-(SEQ ID NO:52)-(SEQ ID NO:84)-(SEQ ID NO:100),

(SEQ ID NO:37)-LL-(SEQ ID NO:53)-(SEQ ID NO:85)-(SEQ ID NO:101),

(SEQ ID NO:38)-LL-(SEQ ID NO:54)-(SEQ ID NO:86)-(SEQ ID NO:102),

(SEQ ID NO:39)-LL-(SEQ ID NO:55)-(SEQ ID NO:87)-(SEQ ID NO:103),

(SEQ ID NO:40)-LL-(SEQ ID NO:56)-(SEQ ID NO:88)-(SEQ ID NO:104),

(SEQ ID NO:41)-LL-(SEQ ID NO:57)-(SEQ ID NO:89)-(SEQ ID NO:105),

(SEQ ID NO:42)-LL-(SEQ ID NO:58)-(SEQ ID NO:90)-(SEQ ID NO:106),

(SEQ ID NO:43)-LL-(SEQ ID NO:59)-(SEQ ID NO:91)-(SEQ ID NO:107),

(SEQ ID NO:44)-LL-(SEQ ID NO:60)-(SEQ ID NO:92)-(SEQ ID NO:108),

(SEQ ID NO:45)-LL-(SEQ ID NO:61)-(SEQ ID NO:93)-(SEQ ID NO:109),

(SEQ ID NO:46)-LL-(SEQ ID NO:62)-(SEQ ID NO:94)-(SEQ ID NO:110),

(SEQ ID NO:47)-LL-(SEQ ID NO:63)-(SEQ ID NO:95)-(SEQ ID NO:111),

(SEQ ID NO:48)-LL-(SEQ ID NO:64)-(SEQ ID NO:96)-(SEQ ID NO:112), and

(SEQ ID NO:49)-LL-(SEQ ID NO:65)-(SEQ ID NO:97)-(SEQ ID NO:113),

wherein each sequence above is linked to the adjacent sequence asdescribed herein and wherein LL is a linker as described herein. Inparticular, the HA1 C-terminal segments can be covalently ornon-covalently linked to the HA2 domains. In certain embodiments, LL isselected from the group consisting of a direct bond, Gly, Gly-Gly,Gly-Gly-Gly, Gly-Gly-Gly-Gly (SEQ ID NO:319), (Gly)n, Gly-Pro,ITPNGSIPNDKPFQNVNKITYGA (SEQ ID NO:165) and Asn-Ala-Ser.

In certain embodiments, provided herein is an influenza hemagglutininstem domain polypeptide having a sequence selected from the groupconsisting of:

(SEQ ID NO:34)-LL-(SEQ ID NO:50)-(SEQ ID NO:82)-(SEQ ID NO:168)-(SEQ IDNO:167)-(SEQ ID NO:166),

(SEQ ID NO:35)-LL-(SEQ ID NO:51)-(SEQ ID NO:83)-(SEQ ID NO:168)-(SEQ IDNO:167)-(SEQ ID NO:166),

(SEQ ID NO:36)-LL-(SEQ ID NO:52)-(SEQ ID NO:84)-(SEQ ID NO:168)-(SEQ IDNO:167)-(SEQ ID NO:166),

(SEQ ID NO:37)-LL-(SEQ ID NO:53)-(SEQ ID NO:85)-(SEQ ID NO:168)-(SEQ IDNO:167)-(SEQ ID NO:166),

(SEQ ID NO:38)-LL-(SEQ ID NO:54)-(SEQ ID NO:86)-(SEQ ID NO:168)-(SEQ IDNO:167)-(SEQ ID NO:166),

(SEQ ID NO:39)-LL-(SEQ ID NO:55)-(SEQ ID NO:87)-(SEQ ID NO:168)-(SEQ IDNO:167)-(SEQ ID NO:166),

(SEQ ID NO:40)-LL-(SEQ ID NO:56)-(SEQ ID NO:88)-(SEQ ID NO:168)-(SEQ IDNO:167)-(SEQ ID NO:166),

(SEQ ID NO:41)-LL-(SEQ ID NO:57)-(SEQ ID NO:89)-(SEQ ID NO:168)-(SEQ IDNO:167)-(SEQ ID NO:166),

(SEQ ID NO:42)-LL-(SEQ ID NO:58)-(SEQ ID NO:90)-(SEQ ID NO:168)-(SEQ IDNO:167)-(SEQ ID NO:166),

(SEQ ID NO:43)-LL-(SEQ ID NO:59)-(SEQ ID NO:91)-(SEQ ID NO:168)-(SEQ IDNO:167)-(SEQ ID NO:166),

(SEQ ID NO:44)-LL-(SEQ ID NO:60)-(SEQ ID NO:92)-(SEQ ID NO:168)-(SEQ IDNO:167)-(SEQ ID NO:166),

(SEQ ID NO:45)-LL-(SEQ ID NO:61)-(SEQ ID NO:93)-(SEQ ID NO:168)-(SEQ IDNO:167)-(SEQ ID NO:166),

(SEQ ID NO:46)-LL-(SEQ ID NO:62)-(SEQ ID NO:94)-(SEQ ID NO:168)-(SEQ IDNO:167)-(SEQ ID NO:166),

(SEQ ID NO:47)-LL-(SEQ ID NO:63)-(SEQ ID NO:95)-(SEQ ID NO:168)-(SEQ IDNO:167)-(SEQ ID NO:166),

(SEQ ID NO:48)-LL-(SEQ ID NO:64)-(SEQ ID NO:96)-(SEQ ID NO:168)-(SEQ IDNO:167)-(SEQ ID NO:166), and

(SEQ ID NO:49)-LL-(SEQ ID NO:65)-(SEQ ID NO:97)-(SEQ ID NO:168)-(SEQ IDNO:167)-(SEQ ID NO:166),

wherein each sequence above is linked to the adjacent sequence asdescribed herein and wherein LL is a linker as described herein. Inparticular, the HA1 C-terminal segments can be covalently ornon-covalently linked to the HA2 domains. In certain embodiments, LL isselected from the group consisting of a direct bond, Gly, Gly-Gly,Gly-Gly-Gly, Gly-Gly-Gly-Gly (SEQ ID NO:319), (Gly)n, Gly-Pro,ITPNGSIPNDKPFQNVNKITYGA (SEQ ID NO:165) and Asn-Ala-Ser.

In certain embodiments, provided herein is an influenza hemagglutininstem domain polypeptide having a sequence selected from the groupconsisting of:

(SEQ ID NO:34)-LL-(SEQ ID NO:50)-(SEQ ID NO:82)-(SEQ ID NO:98)-(SEQ IDNO:168)-(SEQ ID NO:167)-(SEQ ID NO:166),

(SEQ ID NO:35)-LL-(SEQ ID NO:51)-(SEQ ID NO:83)-(SEQ ID NO:99)-(SEQ IDNO:168)-(SEQ ID NO:167)-(SEQ ID NO:166),

(SEQ ID NO:36)-LL-(SEQ ID NO:52)-(SEQ ID NO:84)-(SEQ ID NO:100)-(SEQ IDNO:168)-(SEQ ID NO:167)-(SEQ ID NO:166),

(SEQ ID NO:37)-LL-(SEQ ID NO:53)-(SEQ ID NO:85)-(SEQ ID NO:101)-(SEQ IDNO:168)-(SEQ ID NO:167)-(SEQ ID NO:166),

(SEQ ID NO:38)-LL-(SEQ ID NO:54)-(SEQ ID NO:86)-(SEQ ID NO:102)-(SEQ IDNO:168)-(SEQ ID NO:167)-(SEQ ID NO:166),

(SEQ ID NO:39)-LL-(SEQ ID NO:55)-(SEQ ID NO:87)-(SEQ ID NO:103)-(SEQ IDNO:168)-(SEQ ID NO:167)-(SEQ ID NO:166),

(SEQ ID NO:40)-LL-(SEQ ID NO:56)-(SEQ ID NO:88)-(SEQ ID NO:104)-(SEQ IDNO:168)-(SEQ ID NO:167)-(SEQ ID NO:166),

(SEQ ID NO:41)-LL-(SEQ ID NO:57)-(SEQ ID NO:89)-(SEQ ID NO:105)-(SEQ IDNO:168)-(SEQ ID NO:167)-(SEQ ID NO:166),

(SEQ ID NO:42)-LL-(SEQ ID NO:58)-(SEQ ID NO:90)-(SEQ ID NO:106)-(SEQ IDNO:168)-(SEQ ID NO:167)-(SEQ ID NO:166),

(SEQ ID NO:43)-LL-(SEQ ID NO:59)-(SEQ ID NO:91)-(SEQ ID NO:107)-(SEQ IDNO:168)-(SEQ ID NO:167)-(SEQ ID NO:166),

(SEQ ID NO:44)-LL-(SEQ ID NO:60)-(SEQ ID NO:92)-(SEQ ID NO:108)-(SEQ IDNO:168)-(SEQ ID NO:167)-(SEQ ID NO:166),

(SEQ ID NO:45)-LL-(SEQ ID NO:61)-(SEQ ID NO:93)-(SEQ ID NO:109)-(SEQ IDNO:168)-(SEQ ID NO:167)-(SEQ ID NO:166),

(SEQ ID NO:46)-LL-(SEQ ID NO:62)-(SEQ ID NO:94)-(SEQ ID NO:110)-(SEQ IDNO:168)-(SEQ ID NO:167)-(SEQ ID NO:166),

(SEQ ID NO:47)-LL-(SEQ ID NO:63)-(SEQ ID NO:95)-(SEQ ID NO:111)-(SEQ IDNO:168)-(SEQ ID NO:167)-(SEQ ID NO:166),

(SEQ ID NO:48)-LL-(SEQ ID NO:64)-(SEQ ID NO:96)-(SEQ ID NO:112)-(SEQ IDNO:168)-(SEQ ID NO:167)-(SEQ ID NO:166), and

(SEQ ID NO:49)-LL-(SEQ ID NO:65)-(SEQ ID NO:97)-(SEQ ID NO:113)-(SEQ IDNO:168)-(SEQ ID NO:167)-(SEQ ID NO:166),

wherein each sequence above is linked to the adjacent sequence asdescribed herein and wherein LL is a linker as described herein. Inparticular, the HA1 C-terminal segments can be covalently ornon-covalently linked to the HA2 domains. In certain embodiments, LL isselected from the group consisting of a direct bond, Gly, Gly-Gly,Gly-Gly-Gly, Gly-Gly-Gly-Gly (SEQ ID NO:319), Gly-Pro,ITPNGSIPNDKPFQNVNKITYGA (SEQ ID NO:165) and Asn-Ala-Ser.

In certain embodiments, provided herein is an influenza hemagglutininstem domain polypeptide having a sequence selected from the groupconsisting of:

(SEQ ID NO:177)-LL-(SEQ ID NO:226)-(SEQ ID NO:66),

(SEQ ID NO:178)-LL-(SEQ ID NO:227)-(SEQ ID NO:66),

(SEQ ID NO:179)-LL-(SEQ ID NO:228)-(SEQ ID NO:66),

(SEQ ID NO:180)-LL-(SEQ ID NO:229)-(SEQ ID NO:67),

(SEQ ID NO:181)-LL-(SEQ ID NO:230)-(SEQ ID NO:67),

(SEQ ID NO:182)-LL-(SEQ ID NO:231)-(SEQ ID NO:67),

(SEQ ID NO:183)-LL-(SEQ ID NO:232)-(SEQ ID NO:68),

(SEQ ID NO:184)-LL-(SEQ ID NO:233)-(SEQ ID NO:68),

(SEQ ID NO:185)-LL-(SEQ ID NO:234)-(SEQ ID NO:68),

(SEQ ID NO:186)-LL-(SEQ ID NO:235)-(SEQ ID NO:69),

(SEQ ID NO:187)-LL-(SEQ ID NO:236)-(SEQ ID NO:69),

(SEQ ID NO:188)-LL-(SEQ ID NO:237)-(SEQ ID NO:69),

(SEQ ID NO:189)-LL-(SEQ ID NO:238)-(SEQ ID NO:70),

(SEQ ID NO:190)-LL-(SEQ ID NO:239)-(SEQ ID NO:70),

(SEQ ID NO:191)-LL-(SEQ ID NO:240)-(SEQ ID NO:70),

(SEQ ID NO:192)-LL-(SEQ ID NO:241)-(SEQ ID NO:71),

(SEQ ID NO:193)-LL-(SEQ ID NO:242)-(SEQ ID NO:71),

(SEQ ID NO:194)-LL-(SEQ ID NO:243)-(SEQ ID NO:71),

(SEQ ID NO:195)-LL-(SEQ ID NO:244)-(SEQ ID NO:72),

(SEQ ID NO:196)-LL-(SEQ ID NO:245)-(SEQ ID NO:72),

(SEQ ID NO:197)-LL-(SEQ ID NO:246)-(SEQ ID NO:72),

(SEQ ID NO:198)-LL-(SEQ ID NO:247)-(SEQ ID NO:73),

(SEQ ID NO:199)-LL-(SEQ ID NO:248)-(SEQ ID NO:73),

(SEQ ID NO:200)-LL-(SEQ ID NO:249)-(SEQ ID NO:73),

(SEQ ID NO:201)-LL-(SEQ ID NO:250)-(SEQ ID NO:74),

(SEQ ID NO:202)-LL-(SEQ ID NO:251)-(SEQ ID NO:74),

(SEQ ID NO:203)-LL-(SEQ ID NO:252)-(SEQ ID NO:74),

(SEQ ID NO:204)-LL-(SEQ ID NO:253)-(SEQ ID NO:75),

(SEQ ID NO:205)-LL-(SEQ ID NO:254)-(SEQ ID NO:75),

(SEQ ID NO:206)-LL-(SEQ ID NO:255)-(SEQ ID NO:75),

(SEQ ID NO:207)-LL-(SEQ ID NO:256)-(SEQ ID NO:76),

(SEQ ID NO:208)-LL-(SEQ ID NO:257)-(SEQ ID NO:76),

(SEQ ID NO:209)-LL-(SEQ ID NO:258)-(SEQ ID NO:76),

(SEQ ID NO:210)-LL-(SEQ ID NO:259)-(SEQ ID NO:77),

(SEQ ID NO:211)-LL-(SEQ ID NO:260)-(SEQ ID NO:77),

(SEQ ID NO:212)-LL-(SEQ ID NO:261)-(SEQ ID NO:77),

(SEQ ID NO:213)-LL-(SEQ ID NO:262)-(SEQ ID NO:78),

(SEQ ID NO:214)-LL-(SEQ ID NO:263)-(SEQ ID NO:78),

(SEQ ID NO:215)-LL-(SEQ ID NO:264)-(SEQ ID NO:78),

(SEQ ID NO:216)-LL-(SEQ ID NO:265)-(SEQ ID NO:79),

(SEQ ID NO:217)-LL-(SEQ ID NO:266)-(SEQ ID NO:79),

(SEQ ID NO:218)-LL-(SEQ ID NO:267)-(SEQ ID NO:79),

(SEQ ID NO:219)-LL-(SEQ ID NO:268)-(SEQ ID NO:80),

(SEQ ID NO:220)-LL-(SEQ ID NO:269)-(SEQ ID NO:80),

(SEQ ID NO:221)-LL-(SEQ ID NO:270)-(SEQ ID NO:80),

(SEQ ID NO:222)-LL-(SEQ ID NO:271)-(SEQ ID NO:81),

(SEQ ID NO:223)-LL-(SEQ ID NO:272)-(SEQ ID NO:81),

(SEQ ID NO:224)-LL-(SEQ ID NO:273)-(SEQ ID NO:81),

(SEQ ID NO:309)-LL-(SEQ ID NO:310)-(SEQ ID NO:66), and

(SEQ ID NO:308)-LL-(SEQ ID NO:52)-(SEQ ID NO:68),

(wherein each sequence above is linked to the adjacent sequence asdescribed herein and wherein LL is a linker as described herein. Inparticular, the HA1 C-terminal segments can be covalently ornon-covalently linked to the HA2 domains. In certain embodiments, LL isselected from the group consisting of a direct bond, Gly, Gly-Gly,Gly-Gly-Gly, Gly-Gly-Gly-Gly (SEQ ID NO:319), (Gly)n, Gly-Pro,ITPNGSIPNDKPFQNVNKITYGA (SEQ ID NO:165) and Asn-Ala-Ser.

In certain embodiments, provided herein is an influenza hemagglutininstem domain polypeptide having a sequence selected from the groupconsisting of:

(SEQ ID NO:154)-LL-(SEQ ID NO:158)-(SEQ ID NO:160),

(SEQ ID NO:155)-LL-(SEQ ID NO:159)-(SEQ ID NO:160),

(SEQ ID NO:156)-LL-(SEQ ID NO:158)-(SEQ ID NO:160), and

(SEQ ID NO:157)-LL-(SEQ ID NO:159)-(SEQ ID NO:160),

wherein each sequence above is linked to the adjacent sequence asdescribed herein and wherein LL is a linker as described herein. Inparticular, the HA1 C-terminal segments can be covalently ornon-covalently linked to the HA2 domains. In certain embodiments, LL isselected from the group consisting of a direct bond, Gly, Gly-Gly,Gly-Gly-Gly, Gly-Gly-Gly-Gly (SEQ ID NO:319), (Gly)n, Gly-Pro,ITPNGSIPNDKPFQNVNKITYGA (SEQ ID NO:165) and Asn-Ala-Ser.

In certain embodiments, provided herein is an influenza hemagglutininstem domain polypeptide having a sequence selected from the groupconsisting of:

(SEQ ID NO:154)-LL-(SEQ ID NO:158)-(SEQ ID NO:161),

(SEQ ID NO:155)-LL-(SEQ ID NO:159)-(SEQ ID NO:161),

(SEQ ID NO:156)-LL-(SEQ ID NO:158)-(SEQ ID NO:161), and

(SEQ ID NO:157)-LL-(SEQ ID NO:159)-(SEQ ID NO:161),

wherein each sequence above is linked to the adjacent sequence asdescribed herein and wherein LL is a linker as described herein. Inparticular, the HA1 C-terminal segments can be covalently ornon-covalently linked to the HA2 domains. In certain embodiments, LL isselected from the group consisting of a direct bond, Gly, Gly-Gly,Gly-Gly-Gly, Gly-Gly-Gly-Gly (SEQ ID NO:319), (Gly)n, Gly-Pro,ITPNGSIPNDKPFQNVNKITYGA (SEQ ID NO:165) and Asn-Ala-Ser.

In certain embodiments, provided herein is an influenza hemagglutininstem domain polypeptide having a sequence selected from the groupconsisting of:

(SEQ ID NO:154)-LL-(SEQ ID NO:158)-(SEQ ID NO:161)-(SEQ ID NO:162),

(SEQ ID NO:155)-LL-(SEQ ID NO:159)-(SEQ ID NO:161)-(SEQ ID NO:162),

(SEQ ID NO:156)-LL-(SEQ ID NO:158)-(SEQ ID NO:161)-(SEQ ID NO:162), and

(SEQ ID NO:157)-LL-(SEQ ID NO:159)-(SEQ ID NO:161)-(SEQ ID NO:162),

wherein each sequence above is linked to the adjacent sequence asdescribed herein and wherein LL is a linker as described herein. Inparticular, the HA1 C-terminal segments can be covalently ornon-covalently linked to the HA2 domains. In certain embodiments, LL isselected from the group consisting of a direct bond, Gly, Gly-Gly,Gly-Gly-Gly, Gly-Gly-Gly-Gly (SEQ ID NO:319), (Gly)n, Gly-Pro,ITPNGSIPNDKPFQNVNKITYGA (SEQ ID NO:165) and Asn-Ala-Ser.

In certain embodiments, provided herein is an influenza hemagglutininstem domain polypeptide having a sequence selected from the groupconsisting of:

(SEQ ID NO:154)-LL-(SEQ ID NO:158)-(SEQ ID NO:161)-(SEQ ID NO:168)-(SEQID NO:167)-SEQ ID NO:166),

(SEQ ID NO:155)-LL-(SEQ ID NO:159)-(SEQ ID NO:161)-(SEQ ID NO:168)-(SEQID NO:167)-SEQ ID NO:166),

(SEQ ID NO:156)-LL-(SEQ ID NO:158)-(SEQ ID NO:161)-(SEQ ID NO:168)-(SEQID NO:167)-SEQ ID NO:166), and

(SEQ ID NO:157)-LL-(SEQ ID NO:159)-(SEQ ID NO:161)-(SEQ ID NO:168)-(SEQID NO:167)-SEQ ID NO:166),

wherein each sequence above is linked to the adjacent sequence asdescribed herein and wherein LL is a linker as described herein. Inparticular, the HA1 C-terminal segments can be covalently ornon-covalently linked to the HA2 domains. In certain embodiments, LL isselected from the group consisting of a direct bond, Gly, Gly-Gly,Gly-Gly-Gly, Gly-Gly-Gly-Gly (SEQ ID NO:319), (Gly)n, Gly-Pro,ITPNGSIPNDKPFQNVNKITYGA (SEQ ID NO:165) and Asn-Ala-Ser.

In certain embodiments, provided herein is an influenza hemagglutininstem domain polypeptide having a sequence selected from the groupconsisting of:

(SEQ ID NO:154)-LL-(SEQ ID NO:158)-(SEQ ID NO:161)-(SEQ ID NO:162)-(SEQID NO:168)-(SEQ ID NO:167)-SEQ ID NO:166),

(SEQ ID NO:155)-LL-(SEQ ID NO:159)-(SEQ ID NO:161)-(SEQ ID NO:162)-(SEQID NO:168)-(SEQ ID NO:167)-SEQ ID NO:166),

(SEQ ID NO:156)-LL-(SEQ ID NO:158)-(SEQ ID NO:161)-(SEQ ID NO:162)-(SEQID NO:168)-(SEQ ID NO:167)-SEQ ID NO:166), and

(SEQ ID NO:157)-LL-(SEQ ID NO:159)-(SEQ ID NO:161)-(SEQ ID NO:162)-(SEQID NO:168)-(SEQ ID NO:167)-SEQ ID NO:166),

wherein each sequence above is linked to the adjacent sequence asdescribed herein and wherein LL is a linker as described herein. Inparticular, the HA1 C-terminal segments can be covalently ornon-covalently linked to the HA2 domains. In certain embodiments, LL isselected from the group consisting of a direct bond, Gly, Gly-Gly,Gly-Gly-Gly, Gly-Gly-Gly-Gly (SEQ ID NO:319), (Gly)n, Gly-Pro,ITPNGSIPNDKPFQNVNKITYGA (SEQ ID NO:165) and Asn-Ala-Ser.

In certain embodiments, the influenza hemagglutinin polypeptidesdescribed herein do not comprise polypeptides having the amino acidsequence of either Thr-Gly-Leu-Arg-Asn (SEQ ID NO:313) orGly-Ile-Thr-Asn-Lys-Val-Asn-Ser-Val-Ile-Glu-Lys (SEQ ID NO:314). Incertain embodiments, the influenza hemagglutinin polypeptides describedherein do not comprise polypeptides having the amino acid sequence ofThr-Gly-Leu-Arg-Asn (SEQ ID NO:313) andGly-Ile-Thr-Asn-Lys-Val-Asn-Ser-Val-Ile-Glu-Lys (SEQ ID NO:314). Incertain embodiments, the influenza hemagglutinin polypeptides describedherein do not comprise polypeptides having the amino acid sequence ofeither Thr-Gly-Met-Arg-Asn (SEQ ID NO:316) orGln-Ile-Asn-Gly-Lys-Leu-Asn-Arg-Leu-Ile-Glu-Lys (SEQ ID NO:317). Incertain embodiments, the influenza hemagglutinin polypeptides describedherein do not comprise polypeptides having the amino acid sequence ofThr-Gly-Met-Arg-Asn (SEQ ID NO:316) andGln-Ile-Asn-Gly-Lys-Leu-Asn-Arg-Leu-Ile-Glu-Lys (SEQ ID NO:317). Incertain embodiments, the influenza hemagglutinin polypeptides describedherein do not comprise polypeptides having the amino acid sequence ofeither Thr-Gly-Met-Arg-Asn (SEQ ID NO:316) orGln-Ile-Asn-Gly-Lys-Leu-Asn-Arg-Val-Ile-Glu-Lys (SEQ ID NO:318). Incertain embodiments, the influenza hemagglutinin polypeptides describedherein do not comprise polypeptides having the amino acid sequence ofThr-Gly-Met-Arg-Asn (SEQ ID NO:316) andGln-Ile-Asn-Gly-Lys-Leu-Asn-Arg-Val-Ile-Glu-Lys (SEQ ID NO:318).

In certain embodiments, the influenza hemagglutinin polypeptidesdescribed herein are not recognized or bound by the antibody C179(produced by hybridoma FERM BP-4517; clones sold by Takara Bio, Inc.(Otsu, Shiga, Japan)) or by the antibody AI3C (FERM BP-4516).

5.2 Nucleic Acids Encoding Influenza Hemagglutinin Stem DomainPolypeptides

Provided herein are nucleic acids that encode an influenza hemagglutininstem domain polypeptide. In a specific embodiment, provided herein is anucleic acid that encodes an influenza virus hemagglutinin stem domainpolypeptide. Due to the degeneracy of the genetic code, any nucleic acidthat encodes an influenza hemagglutinin stem domain polypeptidedescribed herein is encompassed herein. In certain embodiments, nucleicacids corresponding to naturally occurring influenza virus nucleic acidsencoding an HA1 N-terminal stem segment, an HA1 C-terminal stem segment,HA2 domain, luminal domain, transmembrane domain, and/or cytoplasmicdomain are used to produce an influenza hemagglutinin stem domainpolypeptide.

Also provided herein are nucleic acids capable of hybridizing to anucleic acid encoding an influenza hemagglutinin stem domainpolypeptide. In certain embodiments, provided herein are nucleic acidscapable of hybridizing to a fragment of a nucleic acid encoding aninfluenza hemagglutinin stem domain polypeptide. In other embodiments,provided herein are nucleic acids capable of hybridizing to the fulllength of a nucleic acid encoding an influenza hemagglutinin stem domainpolypeptide. General parameters for hybridization conditions for nucleicacids are described in Sambrook et al., Molecular Cloning—A LaboratoryManual (2nd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y. (1989), and in Ausubel et al., Current Protocols inMolecular Biology, vol. 2, Current Protocols Publishing, New York(1994). Hybridization may be performed under high stringency conditions,medium stringency conditions, or low stringency conditions. Those ofskill in the art will understand that low, medium and high stringencyconditions are contingent upon multiple factors all of which interactand are also dependent upon the nucleic acids in question. For example,high stringency conditions may include temperatures within 5° C. meltingtemperature of the nucleic acid(s), a low salt concentration (e.g., lessthan 250 mM), and a high co-solvent concentration (e.g., 1-20% ofco-solvent, e.g., DMSO). Low stringency conditions, on the other hand,may include temperatures greater than 10° C. below the meltingtemperature of the nucleic acid(s), a high salt concentration (e.g.,greater than 1000 mM) and the absence of co-solvents.

In some embodiments, a nucleic acid encoding an influenza virushemagglutinin stem domain polypeptide is isolated. In certainembodiments, an “isolated” nucleic acid refers to a nucleic acidmolecule which is separated from other nucleic acid molecules which arepresent in the natural source of the nucleic acid. In other words, theisolated nucleic acid can comprise heterologous nucleic acids that arenot associated with it in nature. In other embodiments, an “isolated”nucleic acid, such as a cDNA molecule, can be substantially free ofother cellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized. The term “substantially free ofcellular material” includes preparations of nucleic acid in which thenucleic acid is separated from cellular components of the cells fromwhich it is isolated or recombinantly produced. Thus, nucleic acid thatis substantially free of cellular material includes preparations ofnucleic acid having less than about 30%, 20%, 10%, or 5% (by dry weight)of other nucleic acids. The term “substantially free of culture medium”includes preparations of nucleic acid in which the culture mediumrepresents less than about 50%, 20%, 10%, or 5% of the volume of thepreparation. The term “substantially free of chemical precursors orother chemicals” includes preparations in which the nucleic acid isseparated from chemical precursors or other chemicals which are involvedin the synthesis of the nucleic acid. In specific embodiments, suchpreparations of the nucleic acid have less than about 50%, 30%, 20%,10%, 5% (by dry weight) of chemical precursors or compounds other thanthe nucleic acid of interest.

In addition, provided herein are nucleic acids encoding the individualcomponents of an influenza hemagglutinin stem domain polypeptide. Inspecific embodiments, nucleic acids encoding an HA1 N-terminal stemsegment, an HA1 C-terminal stem segment and/or HA2 domain are provided.Nucleic acids encoding components of an influenza hemagglutinin stemdomain polypeptide may be assembled using standard molecular biologytechniques known to the one of skill in the art.

5.3 Expression of Influenza Hemagglutinin Stem Domain Polypeptides

Provided herein are vectors, including expression vectors, containing anucleic acid encoding an influenza hemagglutinin stem domainpolypeptide. In a specific embodiment, the vector is an expressionvector that is capable of directing the expression of a nucleic acidencoding an influenza hemagglutinin stem domain polypeptide.Non-limiting examples of expression vectors include, but are not limitedto, plasmids and viral vectors, such as replication defectiveretroviruses, adenoviruses, adeno-associated viruses and baculoviruses.

In some embodiments, provided herein are expression vectors encodingcomponents of an influenza hemagglutinin stem domain polypeptide (e.g.,HA1 N-terminal stem segment, an HA1 C-terminal stem segment and/or anHA2). Such vectors may be used to express the components in one or morehost cells and the components may be isolated and conjugated togetherwith a linker using techniques known to one of skill in the art.

An expression vector comprises a nucleic acid encoding an influenzahemagglutinin stem domain polypeptide in a form suitable for expressionof the nucleic acid in a host cell. In a specific embodiment, anexpression vector includes one or more regulatory sequences, selected onthe basis of the host cells to be used for expression, which is operablylinked to the nucleic acid to be expressed. Within an expression vector,“operably linked” is intended to mean that a nucleic acid of interest islinked to the regulatory sequence(s) in a manner which allows forexpression of the nucleic acid (e.g., in an in vitrotranscription/translation system or in a host cell when the vector isintroduced into the host cell). Regulatory sequences include promoters,enhancers and other expression control elements (e.g., polyadenylationsignals). Regulatory sequences include those which direct constitutiveexpression of a nucleic acid in many types of host cells, those whichdirect expression of the nucleic acid only in certain host cells (e.g.,tissue-specific regulatory sequences), and those which direct theexpression of the nucleic acid upon stimulation with a particular agent(e.g., inducible regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed, thelevel of expression of protein desired, etc. The term “host cell” isintended to include a particular subject cell transformed or transfectedwith a nucleic acid and the progeny or potential progeny of such a cell.Progeny of such a cell may not be identical to the parent celltransformed or transfected with the nucleic acid due to mutations orenvironmental influences that may occur in succeeding generations orintegration of the nucleic acid into the host cell genome.

Expression vectors can be designed for expression of an influenzahemagglutinin stem domain polypeptide using prokaryotic (e g., E. coli)or eukaryotic cells (e.g., insect cells (using baculovirus expressionvectors, see, e.g., Treanor et al., 2007, JAMA, 297(14):1577-1582incorporated by reference herein in its entirety), yeast cells, plantcells, algae or mammalian cells). Examples of mammalian host cellsinclude, but are not limited to, Crucell Per.C6 cells, Vero cells, CHOcells, VERY cells, BHK cells, HeLa cells, COS cells, MDCK cells, 293cells, 3T3 cells or WI38 cells. In certain embodiments, the hosts cellsare myeloma cells, e.g., NS0 cells, 45.6 TG1.7 cells, AF-2 clone 9B5cells, AF-2 clone 9B5 cells, J558L cells, MOPC 315 cells, MPC-11 cells,NCI-H929 cells, NP cells, NS0/1 cells, P3 NS1 Ag4 cells, P3/NS1/1-Ag4-1cells, P3U1 cells, P3X63Ag8 cells, P3X63Ag8.653 cells, P3X63Ag8U.1cells, RPMI 8226 cells, Sp20-Ag14 cells, U266B1 cells, X63AG8.653 cells,Y3.Ag.1.2.3 cells, and YO cells. Non-limiting examples of insect cellsinclude Sf9, Sf21, Trichoplusia ni, Spodoptera frugiperda and Bombyxmori. In a particular embodiment, a mammalian cell culture system (e.g.Chinese hamster ovary or baby hamster kidney cells) is used forexpression of an influenza hemagglutinin stem domain polypeptide. Inanother embodiment, a plant cell culture sytem is used for expression ofan influenza hemagglutinin stem domain polypeptide. See, e.g., U.S. Pat.Nos. 7,504,560; 6,770,799; 6,551,820; 6,136,320; 6,034,298; 5,914,935;5,612,487; and 5,484,719, and U.S. patent application publication Nos.2009/0208477, 2009/0082548, 2009/0053762, 2008/0038232, 2007/0275014 and2006/0204487 for plant cells and methods for the production of proteinsutilizing plant cell culture systems.

An expression vector can be introduced into host cells via conventionaltransformation or transfection techniques. Such techniques include, butare not limited to, calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection, andelectroporation. Suitable methods for transforming or transfecting hostcells can be found in Sambrook et al., 1989, Molecular Cloning—ALaboratory Manual, 2nd Edition, Cold Spring Harbor Press, New York, andother laboratory manuals. In certain embodiments, a host cell istransiently transfected with an expression vector containing a nucleicacid encoding an influenza hemagglutinin stem domain polypeptide. Inother embodiments, a host cell is stably transfected with an expressionvector containing a nucleic acid encoding an influenza hemagglutininstem domain polypeptide.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a nucleic acid thatencodes a selectable marker (e.g., for resistance to antibiotics) isgenerally introduced into the host cells along with the nucleic acid ofinterest. Examples of selectable markers include those which conferresistance to drugs, such as G418, hygromycin and methotrexate. Cellsstably transfected with the introduced nucleic acid can be identified bydrug selection (e.g., cells that have incorporated the selectable markergene will survive, while the other cells die).

As an alternative to recombinant expression of an influenzahemagglutinin stem domain polypeptide using a host cell, an expressionvector containing a nucleic acid encoding an influenza hemagglutininstem domain polypeptide can be transcribed and translated in vitrousing, e.g., T7 promoter regulatory sequences and T7 polymerase. In aspecific embodiment, a coupled transcription/translation system, such asPromega TNT®, or a cell lysate or cell extract comprising the componentsnecessary for transcription and translation may be used to produce aninfluenza hemagglutinin stem domain polypeptide.

Once an influenza hemagglutinin stem domain polypeptide has beenproduced, it may be isolated or purified by any method known in the artfor isolation or purification of a protein, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigen, by Protein A, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the isolation or purification of proteins.In certain embodiments, an influenza hemagglutinin stem domainpolypeptide may be conjugated to heterologous proteins, e.g., a majorhistocompatibility complex (MEW) with or without heat shock proteins(e.g., Hsp10, Hsp20, Hsp30, Hsp40, Hsp60, Hsp70, Hsp90, or Hsp100). Incertain embodiments, an influenza hemagglutinin stem domain polypeptidemay be conjugated to immunomodulatory molecules, such as proteins whichwould target the influenza hemagglutinin stem domain polypeptide toimmune cells such as B cells (e.g., C3d) or T cells. In certainembodiments, an influenza hemagglutinin stem domain polypeptide may beconjugated to proteins which stimulate the innate immune system such asinterferon type 1, alpha, beta, or gamma interferon, colony stimulatingfactors such as granulocyte-macrophage colony-stimulating factor(GM-CSF), interleukin (IL)-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12,IL-15, IL-18, IL-21, IL-23, tumor necrosis factor (TNF)-β, TNFα, B7.1,B7.2, 4-1BB, CD40 ligand (CD40L), and drug-inducible CD40 (iCD40).

Accordingly, provided herein are methods for producing an influenzahemagglutinin stem domain polypeptide. In one embodiment, the methodcomprises culturing a host cell containing a nucleic acid encoding thepolypeptide in a suitable medium such that the polypeptide is produced.In some embodiments, the method further comprises isolating thepolypeptide from the medium or the host cell.

5.4 Influenza Virus Vectors

In one aspect, provided herein are influenza viruses containing aninfluenza hemagglutinin stem domain polypeptide. In a specificembodiment, the influenza hemagglutinin stem domain polypeptide isincorporated into the virions of the influenza virus. The influenzaviruses may be conjugated to moieties that target the viruses toparticular cell types, such as immune cells. In some embodiments, thevirions of the influenza virus have incorporated into them or express aheterologous polypeptide in addition to an influenza hemagglutinin stemdomain polypeptide. The heterologous polypeptide may be a polypeptidethat has immunopotentiating activity, or that targets the influenzavirus to a particular cell type, such as an antibody that binds to anantigen on a specific cell type or a ligand that binds a specificreceptor on a specific cell type.

Influenza viruses containing an influenza hemagglutinin stem domainpolypeptide may be produced by supplying in trans the influenzahemagglutinin stem domain polypeptide during production of virions usingtechniques known to one skilled in the art, such as reverse genetics andhelper-free plasmid rescue. Alternatively, the replication of a parentalinfluenza virus comprising a genome engineered to express an influenzahemagglutinin stem domain polypeptide in cells susceptible to infectionwith the virus wherein hemagglutinin function is provided in trans willproduce progeny influenza viruses containing the influenza hemagglutininstem domain polypeptide.

In another aspect, provided herein are influenza viruses comprising agenome engineered to express an influenza hemagglutinin stem domainpolypeptide. In a specific embodiment, the genome of a parentalinfluenza virus is engineered to encode an influenza hemagglutinin stemdomain polypeptide, which is expressed by progeny influenza virus. Inanother specific embodiment, the genome of a parental influenza virus isengineered to encode an influenza hemagglutinin stem domain polypeptide,which is expressed and incorporated into the virions of progenyinfluenza virus. Thus, the progeny influenza virus resulting from thereplication of the parental influenza virus contain an influenzahemagglutinin stem domain polypeptide. The virions of the parentalinfluenza virus may have incorporated into them an influenza virushemagglutinin polypeptide that is from the same or a different type,subtype or strain of influenza virus. Alternatively, the virions of theparental influenza virus may have incorporated into them a moiety thatis capable of functionally replacing one or more of the activities ofinfluenza virus hemagglutinin polypeptide (e.g., the receptor bindingand/or fusogenic activities of influenza virus hemagglutinin). Incertain embodiments, one or more of the activities of the influenzavirus hemagglutinin polypeptide is provided by a fusion proteincomprising (i) an ectodomain of a polypeptide heterologous to influenzavirus fused to (ii) a transmembrane domain, or a transmembrane domainand a cytoplasmic domain of an influenza virus hemagglutininpolypeptide. In a specific embodiment, the virions of the parentalinfluenza virus may have incorporated into them a fusion proteincomprising (i) an ectodomain of a receptor binding/fusogenic polypeptideof an infectious agent other than influenza virus fused to (ii) atransmembrane domain, or a transmembrane domain and a cytoplasmic domainof an influenza virus hemagglutinin. For a description of fusionproteins that provide one or more activities of an influenza virushemagglutinin polypeptide and methods for the production of influenzaviruses engineered to express such fusion proteins, see, e.g.,International patent application Publication No. WO 2007/064802,published Jun. 7, 2007, which is incorporated herein by reference in itsentirety.

In some embodiments, the virions of the parental influenza virus haveincorporated into them a heterologous polypeptide. In certainembodiments, the genome of a parental influenza virus is engineered toencode a heterologous polypeptide and an influenza virus hemagglutininstem domain polypeptide, which are expressed by progeny influenza virus.In specific embodiments, the influenza hemagglutinin stem domainpolypeptide, the heterologous polypeptide or both are incorporated intovirions of the progeny influenza virus.

The heterologous polypeptide may be a polypeptide that targets theinfluenza virus to a particular cell type, such as an antibody thatrecognizes an antigen on a specific cell type or a ligand that binds aspecific receptor on a specific cell type. In some embodiments, thetargeting polypeptide replaces the target cell recognition function ofthe virus. In a specific embodiment, the heterologous polypeptidetargets the influenza virus to the same cell types that influenza virusinfects in nature. In other specific embodiments, the heterologouspolypeptide targets the progeny influenza virus to immune cells, such asB cells, T cells, macrophages or dendritic cells. In some embodiments,the heterologous polypeptide recognizes and binds to cell-specificmarkers of antigen presenting cells, such as dendritic cells (e.g., suchas CD44). In one embodiment, the heterologous polypeptide is DC-SIGNwhich targets the virus to dendritic cells. In another embodiment, theheterologous polypeptide is an antibody (e.g., a single-chain antibody)that targets the virus to an immune cell, which may be fused with atransmembrane domain from another polypeptide so that it is incorporatedinto the influenza virus virion. In some embodiments, the antibody is aCD20 antibody, a CD34 antibody, or an antibody against December-205.Techniques for engineering viruses to express polypeptides withtargeting functions are known in the art. See, e.g., Yang et al., 2006,PNAS 103: 11479-11484 and United States patent application PublicationNo. 20080019998, published Jan. 24, 2008, and No. 20070020238, publishedJan. 25, 2007, the contents of each of which are incorporated herein intheir entirety.

In another embodiment, the heterologous polypeptide is a viralattachment protein. Non-limiting examples of viruses whose attachmentprotein(s) can be used in this aspect are viruses selected from thegroup of: Lassa fever virus, Hepatitis B virus, Rabies virus, Newcastledisease virus (NDV), a retrovirus such as human immunodeficiency virus,tick-borne encephalitis virus, vaccinia virus, herpesvirus, poliovirus,alphaviruses such as Semliki Forest virus, Ross River virus, and Auravirus (which comprise surface glycoproteins such as E1, E2, and E3),Borna disease virus, Hantaan virus, foamyvirus, and SARS-CoV virus.

In one embodiment, a flavivirus surface glycoprotein may be used, suchas Dengue virus (DV) E protein. In some embodiments, a Sindbis virusglycoprotein from the alphavirus family is used (K. S. Wang, R. J. Kuhn,E. G. Strauss, S. Ou, J. H. Strauss, J. Virol. 66, 4992 (1992)). Incertain embodiments, the heterologous polypeptide is derived from an NDVHN or F protein; a human immunodeficiency virus (HIV) gp160 (or aproduct thereof, such as gp41 or gp120); a hepatitis B virus surfaceantigen (HBsAg); a glycoprotein of herpesvirus (e.g., gD, gE); or VP1 ofpoliovirus.

In another embodiment, the heterologous polypeptide is derived from anynon-viral targeting system known in the art. In certain embodiments, aprotein of a nonviral pathogen such as an intracellular bacteria orprotozoa is used. In some embodiments, the bacterial polypeptide isprovided by, e.g., Chlamydia, Rikettsia, Coxelia, Listeria, Brucella, orLegionella. In some embodiments, protozoan polypeptide is provided by,e.g., Plasmodia species, Leishmania spp., Toxoplasma gondii, orTrypanosoma cruzi. Other exemplary targeting systems are described inWaehler et al., 2007, “Engineering targeted viral vectors for genetherapy,” Nature Reviews Genetics 8: 573-587, which is incorporatedherein in its entirety.

In certain embodiments, the heterologous polypeptide expressed by aninfluenza virus has immunopotentiating (immune stimulating) activity.Non-limiting examples of immunopotentiating polypeptides include, butare not limited to, stimulation molecules, cytokines, chemokines,antibodies and other agents such as Flt-3 ligands. Specific examples ofpolypeptides with immunopotentiating activity include: interferon type1, alpha, beta, or gamma interferon, colony stimulating factors such asgranulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin(IL)-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21, IL-23,tumor necrosis factor (TNF)-β, TNFα, B7.1, B7.2, 4-1BB, CD40 ligand(CD40L), and drug-inducible CD40 (iCD40) (see, e.g., Hanks, B. A., etal. 2005. Nat Med 11:130-137, which is incorporated herein by referencein its entirety.)

Since the genome of influenza A and B viruses consist of eight (8)single-stranded, negative sense segments (influenza C viruses consist ofseven (7) single-stranded, negative sense segments), the genome of aparental influenza virus may be engineered to express an influenzahemagglutinin stem domain polypeptide (and any other polypeptide, suchas a heterologous polypeptide) using a recombinant segment andtechniques known to one skilled in the art, such a reverse genetics andhelper-free plasmid rescue. In one embodiment, the recombinant segmentcomprises a nucleic acid encoding the influenza hemagglutinin stemdomain polypeptide as well as the 3′ and 5′ incorporation signals whichare required for proper replication, transcription and packaging of thevRNAs (Fujii et al., 2003, Proc. Natl. Acad. Sci. USA 100:2002-2007;Zheng, et al., 1996, Virology 217:242-251, both of which areincorporated by reference herein in their entireties). In a specificembodiment, the recombinant segment uses the 3′ and 5′ noncoding and/ornontranslated sequences of segments of influenza viruses that are from adifferent or the same type, subtype or strain as the parental influenzavirus. In some embodiments, the recombinant segment comprises the 3′noncoding region of an influenza virus hemagglutinin polypeptide, theuntranslated regions of an influenza virus hemagglutinin polypeptide,and the 5′ non-coding region of an influenza virus hemagglutininpolypeptide. In specific embodiments, the recombinant segment comprisesthe 3′ and 5′ noncoding and/or nontranslated sequences of the HA segmentof an influenza virus that is the same type, subtype or strain as theinfluenza virus type, subtype or strain as the HA1 N-terminal stemsegment, the HA1 C-terminal stem segment and/or the HA2 of an influenzahemagglutinin stem domain polypeptide. In certain embodiments, therecombinant segment encoding the influenza hemagglutinin stem domainpolypeptide may replace the HA segment of a parental influenza virus. Insome embodiments, the recombinant segment encoding the influenzahemagglutinin stem domain polypeptide may replace the NS1 gene of theparental influenza virus. In some embodiments, the recombinant segmentencoding the influenza hemagglutinin stem domain polypeptide may replacethe NA gene of the parental influenza virus. Exemplary influenza virusstrains that can be used to express the influenza hemagglutinin stemdomain polypeptides include Ann Arbor/1/50, A/Puerto Rico/8/34, A/SouthDakota/6/2007, A/Uruguay/716/2007, and B/Brisbane/60/2008.

In some embodiments, the genome of a parental influenza virus may beengineered to express an influenza hemagglutinin stem domain polypeptideusing a recombinant segment that is bicistronic. Bicistronic techniquesallow the engineering of coding sequences of multiple proteins into asingle mRNA through the use of internal ribosome entry site (IRES)sequences. IRES sequences direct the internal recruitment of ribosomesto the RNA molecule and allow downstream translation in a capindependent manner. Briefly, a coding region of one protein is insertedinto the open reading frame (ORF) of a second protein. The insertion isflanked by an IRES and any untranslated signal sequences necessary forproper expression and/or function. The insertion must not disrupt theORF, polyadenylation or transcriptional promoters of the second protein(see, e.g., Garcia-Sastre et al., 1994, J. Virol. 68:6254-6261 andGarcia-Sastre et al., 1994 Dev. Biol. Stand. 82:237-246, each of whichis hereby incorporated by reference in its entirety). See also, e.g.,U.S. Pat. No. 6,887,699, U.S. Pat. No. 6,001,634, U.S. Pat. No.5,854,037 and U.S. Pat. No. 5,820,871, each of which is incorporatedherein by reference in its entirety. Any IRES known in the art ordescribed herein may be used in accordance with the invention (e.g., theIRES of BiP gene, nucleotides 372 to 592 of GenBank database entryHUMGRP78; or the IRES of encephalomyocarditis virus (EMCV), nucleotides1430-2115 of GenBank database entry CQ867238.). Thus, in certainembodiments, a parental influenza virus is engineered to contain abicistronic RNA segment that expresses the influenza hemagglutinin stemdomain polypeptide and another polypeptide, such as gene expressed bythe parental influenza virus. In some embodiments, the parentalinfluenza virus gene is the HA gene. In some embodiments, the parentalinfluenza virus gene is the NA gene. In some embodiments, the parentalinfluenza virus gene is the NS1 gene.

Techniques known to one skilled in the art may be used to produce aninfluenza virus containing an influenza hemagglutinin stem domainpolypeptide and an influenza virus comprising a genome engineered toexpress an influenza hemagglutinin stem domain polypeptide. For example,reverse genetics techniques may be used to generate such an influenzavirus. Briefly, reverse genetics techniques generally involve thepreparation of synthetic recombinant viral RNAs that contain thenon-coding regions of the negative-strand, viral RNA which are essentialfor the recognition by viral polymerases and for packaging signalsnecessary to generate a mature virion. The recombinant RNAs aresynthesized from a recombinant DNA template and reconstituted in vitrowith purified viral polymerase complex to form recombinantribonucleoproteins (RNPs) which can be used to transfect cells. A moreefficient transfection is achieved if the viral polymerase proteins arepresent during transcription of the synthetic RNAs either in vitro or invivo. The synthetic recombinant RNPs can be rescued into infectiousvirus particles. The foregoing techniques are described in U.S. Pat. No.5,166,057 issued Nov. 24, 1992; in U.S. Pat. No. 5,854,037 issued Dec.29, 1998; in European Patent Publication EP 0702085A1, published Feb.20, 1996; in U.S. patent application Ser. No. 09/152,845; inInternational Patent Publications PCT WO 97/12032 published Apr. 3,1997; WO 96/34625 published Nov. 7, 1996; in European Patent PublicationEP A780475; WO 99/02657 published Jan. 21, 1999; WO 98/53078 publishedNov. 26, 1998; WO 98/02530 published Jan. 22, 1998; WO 99/15672published Apr. 1, 1999; WO 98/13501 published Apr. 2, 1998; WO 97/06270published Feb. 20, 1997; and EPO 780 475A1 published Jun. 25, 1997, eachof which is incorporated by reference herein in its entirety.

Alternatively, helper-free plasmid technology may be used to produce aninfluenza virus containing an influenza hemagglutinin stem domainpolypeptide and an influenza virus comprising a genome engineered toexpress an influenza hemagglutinin stem domain polypeptide. Briefly,full length cDNAs of viral segments are amplified using PCR with primersthat include unique restriction sites, which allow the insertion of thePCR product into the plasmid vector (Flandorfer et al., 2003, J. Virol.77:9116-9123; Nakaya et al., 2001, J. Virol. 75:11868-11873; both ofwhich are incorporated herein by reference in their entireties). Theplasmid vector is designed so that an exact negative (vRNA sense)transcript is expressed. For example, the plasmid vector may be designedto position the PCR product between a truncated human RNA polymerase Ipromoter and a hepatitis delta virus ribozyme sequence such that anexact negative (vRNA sense) transcript is produced from the polymerase Ipromoter. Separate plasmid vectors comprising each viral segment as wellas expression vectors comprising necessary viral proteins may betransfected into cells leading to production of recombinant viralparticles. In another example, plasmid vectors from which both the viralgenomic RNA and mRNA encoding the necessary viral proteins are expressedmay be used. For a detailed description of helper-free plasmidtechnology see, e.g., International Publication No. WO 01/04333; U.S.Pat. Nos. 6,951,754, 7,384,774, 6,649,372, and 7,312,064; Fodor et al.,1999, J. Virol. 73:9679-9682; Quinlivan et al., 2005, J. Virol.79:8431-8439; Hoffmann et al., 2000, Proc. Natl. Acad. Sci. USA97:6108-6113; and Neumann et al., 1999, Proc. Natl. Acad. Sci. USA96:9345-9350, which are incorporated herein by reference in theirentireties.

The influenza viruses described herein may be propagated in anysubstrate that allows the virus to grow to titers that permit their usein accordance with the methods described herein. In one embodiment, thesubstrate allows the viruses to grow to titers comparable to thosedetermined for the corresponding wild-type viruses. In certainembodiments, the substrate is one which is biologically relevant to theinfluenza virus or to the virus from which the HA function is derived.In a specific embodiment, an attenuated influenza virus by virtue of,e.g., a mutation in the NS1 gene, may be propagated in an IFN-deficientsubstrate. For example, a suitable IFN-deficient substrate may be onethat is defective in its ability to produce or respond to interferon, oris one which An IFN-deficient substrate may be used for the growth ofany number of viruses which may require interferon-deficient growthenvironment. See, for example, U.S. Pat. No. 6,573,079, issued Jun. 3,2003, U.S. Pat. No. 6,852,522, issued Feb. 8, 2005, and U.S. Pat. No.7,494,808, issued Feb. 24, 2009, the entire contents of each of which isincorporated herein by reference in its entirety.

The influenza viruses described herein may be isolated and purified byany method known to those of skill in the art. In one embodiment, thevirus is removed from cell culture and separated from cellularcomponents, typically by well known clarification procedures, e.g., suchas gradient centrifugation and column chromatography, and may be furtherpurified as desired using procedures well known to those skilled in theart, e.g., plaque assays.

In certain embodiments, the influenza viruses, or influenza viruspolypeptides, genes or genome segments for use as described herein areobtained or derived from an influenza A virus. In certain embodiments,the influenza viruses, or influenza virus polypeptides, genes or genomesegments for use as described herein are obtained or derived from asingle influenza A virus subtype or strain. In other embodiments, theinfluenza viruses, or influenza virus polypeptides, genes or genomesegments for use as described herein are obtained or derived from two ormore influenza A virus subtypes or strains.

In some embodiments, the influenza viruses, or influenza viruspolypeptides, genes or genome segments for use as described herein areobtained or derived from an influenza B virus. In certain embodiments,the influenza viruses, or influenza virus polypeptides, genes or genomesegments for use as described herein are obtained or derived from asingle influenza B virus subtype or strain. In other embodiments, theinfluenza viruses, or influenza virus polypeptides, genes or genomesegments for use as described herein are obtained or derived from two ormore influenza B virus subtypes or strains. In other embodiments, theinfluenza viruses, or influenza virus polypeptides, genes or genomesegments for use as described herein are obtained or derived from acombination of influenza A and influenza B virus subtypes or strains.

In some embodiments, the influenza viruses, or influenza viruspolypeptides, genes or genome segments for use as described herein areobtained or derived from an influenza C virus. In certain embodiments,the influenza viruses, or influenza virus polypeptides, genes or genomesegments for use as described herein are obtained or derived from asingle influenza C virus subtype or strain. In other embodiments, theinfluenza viruses, or influenza virus polypeptides, genes or genomesegments for use as described herein are obtained or derived from two ormore influenza C virus subtypes or strains. In other embodiments, theinfluenza viruses, or influenza virus polypeptides, genes or genomesegments for use as described herein are obtained or derived from acombination of influenza C virus and influenza A virus and/or influenzaB virus subtypes or strains.

Non-limiting examples of influenza A viruses include subtype H10N4,subtype H10N5, subtype H10N7, subtype H10N8, subtype H10N9, subtypeH11N1, subtype H11N13, subtype H11N2, subtype H11N4, subtype H11N6,subtype H11N8, subtype H11N9, subtype H12N1, subtype H12N4, subtypeH12N5, subtype H12N8, subtype H13N2, subtype H13N3, subtype H13N6,subtype H13N7, subtype H14N5, subtype H14N6, subtype H15N8, subtypeH15N9, subtype H16N3, subtype H1N1, subtype H1N2, subtype H1N3, subtypeH1N6, subtype H1N9, subtype H2N1, subtype H2N2, subtype H2N3, subtypeH2N5, subtype H2N7, subtype H2N8, subtype H2N9, subtype H3N1, subtypeH3N2, subtype H3N3, subtype H3N4, subtype H3N5, subtype H3N6, subtypeH3N8, subtype H3N9, subtype H4N1, subtype H4N2, subtype H4N3, subtypeH4N4, subtype H4N5, subtype H4N6, subtype H4N8, subtype H4N9, subtypeH5N1, subtype H5N2, subtype H5N3, subtype H5N4, subtype H5N6, subtypeH5N7, subtype H5N8, subtype H5N9, subtype H6N1, subtype H6N2, subtypeH6N3, subtype H6N4, subtype H6N5, subtype H6N6, subtype H6N7, subtypeH6N8, subtype H6N9, subtype H7N1, subtype H7N2, subtype H7N3, subtypeH7N4, subtype H7N5, subtype H7N7, subtype H7N8, subtype H7N9, subtypeH8N4, subtype H8N5, subtype H9N1, subtype H9N2, subtype H9N3, subtypeH9N5, subtype H9N6, subtype H9N7, subtype H9N8, and subtype H9N9.

Specific examples of strains of influenza A virus include, but are notlimited to: A/sw/Iowa/15/30 (H1N1); A/WSN/33 (H1N1); A/eq/Prague/1/56(H7N7); A/PR/8/34; A/mallard/Potsdam/178-4/83 (H2N2); A/herringgull/DE/712/88 (H16N3); A/sw/Hong Kong/168/1993 (H1N1);A/mallard/Alberta/211/98 (H1N1); A/shorebird/Delaware/168/06 (H16N3);A/sw/Netherlands/25/80 (H1N1); A/sw/Germany/2/81 (H1N1);A/sw/Hannover/1/81 (H1N1); A/sw/Potsdam/1/81 (H1N1); A/sw/Potsdam/15/81(H1N1); A/sw/Potsdam/268/81 (H1N1); A/sw/Finistere/2899/82 (H1N1);A/sw/Potsdam/35/82 (H3N2); A/sw/Cote d'Armor/3633/84 (H3N2);A/sw/Gent/1/84 (H3N2); A/sw/Netherlands/12/85 (H1N1);A/sw/Karrenzien/2/87 (H3N2); A/sw/Schwerin/103/89 (H1N1);A/turkey/Germany/3/91 (H1N1); A/sw/Germany/8533/91 (H1N1);A/sw/Belgium/220/92 (H3N2); A/sw/Gent/V230/92 (H1N1);A/sw/Leipzig/145/92 (H3N2); A/sw/Re220/92 hp (H3N2); A/sw/Bakum/909/93(H3N2); A/sw/Schleswig-Holstein/1/93 (H1N1); A/sw/Scotland/419440/94(H1N2); A/sw/Bakum/5/95 (H1N1); A/sw/Best/5C/96 (H1N1);A/sw/England/17394/96 (H1N2); A/sw/Jena/5/96 (H3N2);A/sw/Oedenrode/7C/96 (H3N2); A/sw/Lohne/1/97 (H3N2); A/sw/Coted'Armor/790/97 (H1N2); A/sw/Bakum/1362/98 (H3N2); A/sw/Italy/1521/98(H1N2); A/sw/Italy/1553-2/98 (H3N2); A/sw/Italy/1566/98 (H1N1);A/sw/Italy/1589/98 (H1N1); A/sw/Bakum/8602/99 (H3N2); A/sw/Cotesd'Armor/604/99 (H1N2); A/sw/Cote d'Armor/1482/99 (H1N1);A/sw/Gent/7625/99 (H1N2); A/Hong Kong/1774/99 (H3N2); A/sw/HongKong/5190/99 (H3N2); A/sw/Hong Kong/5200/99 (H3N2); A/sw/HongKong/5212/99 (H3N2); A/sw/Ille et Villaine/1455/99 (H1N1);A/sw/Italy/1654-1/99 (H1N2); A/sw/Italy/2034/99 (H1N1);A/sw/Italy/2064/99 (H1N2); A/sw/Berlin/1578/00 (H3N2);A/sw/Bakum/1832/00 (H1N2); A/sw/Bakum/1833/00 (H1N2); A/sw/Coted'Armor/800/00 (H1N2); A/sw/Hong Kong/7982/00 (H3N2); A/sw/Italy/1081/00(H1N2); A/sw/Belzig/2/01 (H1N1); A/sw/Belzig/54/01 (H3N2); A/sw/HongKong/9296/01 (H3N2); A/sw/Hong Kong/9745/01 (H3N2); A/sw/Spain/33601/01(H3N2); A/sw/Hong Kong/1144/02 (H3N2); A/sw/Hong Kong/1197/02 (H3N2);A/sw/Spain/39139/02 (H3N2); A/sw/Spain/42386/02 (H3N2);A/Switzerland/8808/2002 (H1N1); A/sw/Bakum/1769/03 (H3N2);A/sw/Bissendorf/IDT1864/03 (H3N2); A/sw/Ehren/IDT2570/03 (H1N2);A/sw/Gescher/IDT2702/03 (H1N2); A/sw/Haselünne/2617/03 hp (H1N1);A/sw/Loningen/IDT2530/03 (H1N2); A/sw/IVD/IDT2674/03 (H1N2);A/sw/Nordkirchen/IDT1993/03 (H3N2); A/sw/Nordwalde/IDT2197/03 (H1N2);A/sw/Norden/IDT2308/03 (H1N2); A/sw/Spain/50047/03 (H1N1);A/sw/Spain/51915/03 (H1N1); A/sw/Vechta/2623/03 (H1N1);A/sw/Visbek/IDT2869/03 (H1N2); A/sw/Waltersdorf/IDT2527/03 (H1N2);A/sw/Damme/IDT2890/04 (H3N2); A/sw/Geldern/IDT2888/04 (H1N1);A/sw/Granstedt/IDT3475/04 (H1N2); A/sw/Greven/IDT2889/04 (H1N1);A/sw/Gudensberg/IDT2930/04 (H1N2); A/sw/Gudensberg/IDT2931/04 (H1N2);A/sw/Lohne/IDT3357/04 (H3N2); A/sw/Nortrup/IDT3685/04 (H1N2);A/sw/Seesen/IDT3055/04 (H3N2); A/sw/Spain/53207/04 (H1N1);A/sw/Spain/54008/04 (H3N2); A/sw/Stolzenau/IDT3296/04 (H1N2);A/sw/Wedel/IDT2965/04 (H1N1); A/sw/Bad Griesbach/IDT4191/05 (H3N2);A/sw/Cloppenburg/IDT4777/05 (H1N2); A/sw/Dötlingen/IDT3780/05 (H1N2);A/sw/Dötlingen/IDT4735/05 (H1N2); A/sw/Egglham/IDT5250/05 (H3N2);A/sw/Harkenblek/IDT4097/05 (H3N2); A/sw/Hertzen/IDT4317/05 (H3N2);A/sw/Krogel/IDT4192/05 (H1N1); A/sw/Laer/IDT3893/05 (H1N1);A/sw/Laer/IDT4126/05 (H3N2); A/sw/Merzen/IDT4114/05 (H3N2);A/sw/Muesleringen-S./IDT4263/05 (H3N2); A/sw/Osterhofen/IDT4004/05(H3N2); A/sw/Sprenge/IDT3805/05 (H1N2); A/sw/Stadtlohn/IDT3853/05(H1N2); A/sw/Voglarn/IDT4096/05 (H1N1); A/sw/Wohlerst/IDT4093/05 (H1N1);A/sw/Bad Griesbach/IDT5604/06 (H1N1); A/sw/Herzlake/IDT5335/06 (H3N2);A/sw/Herzlake/IDT5336/06 (H3N2); A/sw/Herzlake/IDT5337/06 (H3N2); andA/wild boar/Germany/R169/2006 (H3N2).

Other specific examples of strains of influenza A virus include, but arenot limited to: A/Toronto/3141/2009 (H1N1); A/Regensburg/D6/2009 (H1N1);A/Bayern/62/2009 (H1N1); A/Bayern/62/2009 (H1N1); A/Bradenburg/19/2009(H1N1); A/Bradenburg/20/2009 (H1N1); A/Distrito Federal/2611/2009(H1N1); A/Mato Grosso/2329/2009 (H1N1); A/Sao Paulo/1454/2009 (H1N1);A/Sao Paulo/2233/2009 (H1N1); A/Stockholm/37/2009 (H1N1);A/Stockholm/41/2009 (H1N1); A/Stockholm/45/2009 (H1N1);A/swine/Alberta/OTH-33-1/2009 (H1N1); A/swine/Alberta/OTH-33-14/2009(H1N1); A/swine/Alberta/OTH-33-2/2009 (H1N1);A/swine/Alberta/OTH-33-21/2009 (H1N1); A/swine/Alberta/OTH-33-22/2009(H1N1); A/swine/Alberta/OTH-33-23/2009 (H1N1);A/swine/Alberta/OTH-33-24/2009 (H1N1); A/swine/Alberta/OTH-33-25/2009(H1N1); A/swine/Alberta/OTH-33-3/2009 (H1N1);A/swine/Alberta/OTH-33-7/2009 (H1N1); A/Beijing/502/2009 (H1N1);A/Firenze/10/2009 (H1N1); A/Hong Kong/2369/2009 (H1N1); A/Italy/85/2009(H1N1); A/Santo Domingo/572N/2009 (H1N1); A/Catalonia/385/2009 (H1N1);A/Catalonia/386/2009 (H1N1); A/Catalonia/387/2009 (H1N1);A/Catalonia/390/2009 (H1N1); A/Catalonia/394/2009 (H1N1);A/Catalonia/397/2009 (H1N1); A/Catalonia/398/2009 (H1N1);A/Catalonia/399/2009 (H1N1); A/Sao Paulo/2303/2009 (H1N1);A/Akita/1/2009 (H1N1); A/Castro/JXP/2009 (H1N1); A/Fukushima/1/2009(H1N1); A/Israel/276/2009 (H1N1); A/Israel/277/2009 (H1N1);A/Israel/70/2009 (H1N1); A/Iwate/1/2009 (H1N1); A/Iwate/2/2009 (H1N1);A/Kagoshima/1/2009 (H1N1); A/Osaka/180/2009 (H1N1); A/PuertoMontt/Bio87/2009 (H1 N1); A/Sao Paulo/2303/2009 (H1N1); A/Sapporo/1/2009(H1N1); A/Stockholm/30/2009 (H1N1); A/Stockholm/31/2009 (H1N1);A/Stockholm/32/2009 (H1N1); A/Stockholm/33/2009 (H1N1);A/Stockholm/34/2009 (H1N1); A/Stockholm/35/2009 (H1N1);A/Stockholm/36/2009 (H1N1); A/Stockholm/38/2009 (H1N1);A/Stockholm/39/2009 (H1N1); A/Stockholm/40/2009 (H1N1;)A/Stockholm/42/2009 (H1N1); A/Stockholm/43/2009 (H1N1);A/Stockholm/44/2009 (H1N1); A/Utsunomiya/2/2009 (H1N1);A/WRAIR/0573N/2009 (H1N1); and A/Zhejiang/DTID-ZJU01/2009 (H1N1).

Non-limiting examples of influenza B viruses include strain Aichi/5/88,strain Akita/27/2001, strain Akita/5/2001, strain Alaska/16/2000, strainAlaska/1777/2005, strain Argentina/69/2001, strain Arizona/146/2005,strain Arizona/148/2005, strain Bangkok/163/90, strain Bangkok/34/99,strain Bangkok/460/03, strain Bangkok/54/99, strain Barcelona/215/03,strain Beijing/15/84, strain Beijing/184/93, strain Beijing/243/97,strain Beijing/43/75, strain Beijing/5/76, strain Beijing/76/98, strainBelgium/WV106/2002, strain Belgium/WV107/2002, strainBelgium/WV109/2002, strain Belgium/WV114/2002, strainBelgium/WV122/2002, strain Bonn/43, strain Brazil/952/2001, strainBucharest/795/03, strain Buenos Aires/161/00), strain Buenos Aires/9/95,strain Buenos Aires/SW16/97, strain Buenos Aires/VL518/99, strainCanada/464/2001, strain Canada/464/2002, strain Chaco/366/00, strainChaco/R113/00, strain Cheju/303/03, strain Chiba/447/98, strainChongqing/3/2000, strain clinical isolate SA1 Thailand/2002, strainclinical isolate SA10 Thailand/2002, strain clinical isolate SA100Philippines/2002, strain clinical isolate SA101 Philippines/2002, strainclinical isolate SA110 Philippines/2002), strain clinical isolate SA112Philippines/2002, strain clinical isolate SA113 Philippines/2002, strainclinical isolate SA114 Philippines/2002, strain clinical isolate SA2Thailand/2002, strain clinical isolate SA20 Thailand/2002, strainclinical isolate SA38 Philippines/2002, strain clinical isolate SA39Thailand/2002, strain clinical isolate SA99 Philippines/2002, strainCNIC/27/2001, strain Colorado/2597/2004, strain Cordoba/VA418/99, strainCzechoslovakia/16/89, strain Czechoslovakia/69/90, strain Daeku/10/97,strain Daeku/45/97, strain Daeku/47/97, strain Daeku/9/97, strainB/Du/4/78, strain B/Durban/39/98, strain Durban/43/98, strainDurban/44/98, strain B/Durban/52/98, strain Durban/55/98, strainDurban/56/98, strain England/1716/2005, strain England/2054/2005),strain England/23/04, strain Finland/154/2002, strain Finland/159/2002,strain Finland/160/2002, strain Finland/161/2002, strain Finland/162/03,strain Finland/162/2002, strain Finland/162/91, strain Finland/164/2003,strain Finland/172/91, strain Finland/173/2003, strain Finland/176/2003,strain Finland/184/91, strain Finland/188/2003, strain Finland/190/2003,strain Finland/220/2003, strain Finland/WV5/2002, strain Fujian/36/82,strain Geneva/5079/03, strain Genoa/11/02, strain Genoa/2/02, strainGenoa/21/02, strain Genova/54/02, strain Genova/55/02, strainGuangdong/05/94, strain Guangdong/08/93, strain Guangdong/5/94, strainGuangdong/55/89, strain Guangdong/8/93, strain Guangzhou/7/97, strainGuangzhou/86/92, strain Guangzhou/87/92, strain Gyeonggi/592/2005,strain Hannover/2/90, strain Harbin/07/94, strain Hawaii/10/2001, strainHawaii/1990/2004, strain Hawaii/38/2001, strain Hawaii/9/2001, strainHebei/19/94, strain Hebei/3/94), strain Henan/22/97, strainHiroshima/23/2001, strain Hong Kong/110/99, strain Hong Kong/1115/2002,strain Hong Kong/112/2001, strain Hong Kong/123/2001, strain HongKong/1351/2002, strain Hong Kong/1434/2002, strain Hong Kong/147/99,strain Hong Kong/156/99, strain Hong Kong/157/99, strain HongKong/22/2001, strain Hong Kong/22/89, strain Hong Kong/336/2001, strainHong Kong/666/2001, strain Hong Kong/9/89, strain Houston/1/91, strainHouston/1/96, strain Houston/2/96, strain Hunan/4/72, strainIbaraki/2/85, strain ncheon/297/2005, strain India/3/89, strainIndia/77276/2001, strain Israel/95/03, strain Israel/WV187/2002, strainJapan/1224/2005, strain Jiangsu/10/03, strain Johannesburg/1/99, strainJohannesburg/96/01, strain Kadoma/1076/99, strain Kadoma/122/99, strainKagoshima/15/94, strain Kansas/22992/99, strain Khazkov/224/91, strainKobe/1/2002, strain, strain Kouchi/193/99, strain Lazio/1/02, strainLee/40, strain Leningrad/129/91, strain Lissabon/2/90), strain LosAngeles/1/02, strain Lusaka/270/99, strain Lyon/1271/96, strainMalaysia/83077/2001, strain Maputo/1/99, strain Mar del Plata/595/99,strain Maryland/1/01, strain Memphis/1/01, strain Memphis/12/97-MA,strain Michigan/22572/99, strain Mie/1/93, strain Milano/1/01, strainMinsk/318/90, strain Moscow/3/03, strain Nagoya/20/99, strainNanchang/1/00, strain Nashville/107/93, strain Nashville/45/91, strainNebraska/2/01, strain Netherland/801/90, strain Netherlands/429/98,strain New York/1/2002, strain NIB/48/90, strain Ningxia/45/83, strainNorway/1/84, strain Oman/16299/2001, strain Osaka/1059/97, strainOsaka/983/97-V2, strain Oslo/1329/2002, strain Oslo/1846/2002, strainPanama/45/90, strain Paris/329/90, strain Parma/23/02, strainPerth/211/2001, strain Peru/1364/2004, strain Philippines/5072/2001,strain Pusan/270/99, strain Quebec/173/98, strain Quebec/465/98, strainQuebec/7/01, strain Roma/1/03, strain Saga/S172/99, strain Seoul/13/95,strain Seoul/37/91, strain Shangdong/7/97, strain Shanghai/361/2002),strain Shiga/T30/98, strain Sichuan/379/99, strain Singapore/222/79,strain Spain/WV27/2002, strain Stockholm/10/90, strainSwitzerland/5441/90, strain Taiwan/0409/00, strain Taiwan/0722/02,strain Taiwan/97271/2001, strain Tehran/80/02, strain Tokyo/6/98, strainTrieste/28/02, strain Ulan Ude/4/02, strain United Kingdom/34304/99,strain USSR/100/83, strain Victoria/103/89, strain Vienna/1/99, strainWuhan/356/2000, strain WV194/2002, strain Xuanwu/23/82, strainYamagata/1311/2003, strain Yamagata/K500/2001, strain Alaska/12/96,strain GA/86, strain NAGASAKI/1/87, strain Tokyo/942/96, and strainRochester/02/2001.

Non-limiting examples of influenza C viruses include strain Aichi/1/81,strain Ann Arbor/1/50, strain Aomori/74, strain California/78, strainEngland/83, strain Greece/79, strain Hiroshima/246/2000, strainHiroshima/252/2000, strain Hyogo/1/83, strain Johannesburg/66, strainKanagawa/1/76, strain Kyoto/1/79, strain Mississippi/80, strainMiyagi/1/97, strain Miyagi/5/2000, strain Miyagi/9/96, strain Nara/2/85,strain NewJersey/76, strain pig/Beijing/115/81, strain Saitama/3/2000),strain Shizuoka/79, strain Yamagata/2/98, strain Yamagata/6/2000, strainYamagata/9/96, strain BERLIN/1/85, strain ENGLAND/892/8, strain GREATLAKES/1167/54, strain JJ/50, strain PIG/BEIJING/10/81, strainPIG/BEIJING/439/82), strain TAYLOR/1233/47, and strain C/YAMAGATA/10/81.

In certain embodiments, the influenza viruses provided herein have anattenuated phenotype. In specific embodiments, the attenuated influenzavirus is based on influenza A virus. In other embodiments, theattenuated influenza virus is based on influenza B virus. In yet otherembodiments, the attenuated influenza virus is based on influenza Cvirus. In other embodiments, the attenuated influenza virus may comprisegenes or genome segments from one or more strains or subtypes ofinfluenza A, influenza B, and/or influenza C virus. In some embodiments,the attenuated backbone virus comprises genes from an influenza A virusand an influenza B virus.

In specific embodiments, attenuation of influenza virus is desired suchthat the virus remains, at least partially, infectious and can replicatein vivo, but only generate low titers resulting in subclinical levels ofinfection that are non-pathogenic. Such attenuated viruses areespecially suited for embodiments described herein wherein the virus oran immunogenic composition thereof is administered to a subject toinduce an immune response. Attenuation of the influenza virus can beaccomplished according to any method known in the art, such as, e.g.,selecting viral mutants generated by chemical mutagenesis, mutation ofthe genome by genetic engineering, selecting reassortant viruses thatcontain segments with attenuated function, or selecting for conditionalvirus mutants (e.g., cold-adapted viruses). Alternatively, naturallyoccurring attenuated influenza viruses may be used as influenza virusbackbones for the influenza virus vectors.

In one embodiment, an influenza virus may be attenuated, at least inpart, by virtue of substituting the HA gene of the parental influenzavirus with an influenza hemagglutinin stem domain polypeptide describedherein. In some embodiments, an influenza virus may be attenuated, atleast in part, by engineering the influenza virus to express a mutatedNS1 gene that impairs the ability of the virus to antagonize thecellular interferon (IFN) response. Examples of the types of mutationsthat can be introduced into the influenza virus NS1 gene includedeletions, substitutions, insertions and combinations thereof. One ormore mutations can be introduced anywhere throughout the NS1 gene (e.g.,the N-terminus, the C-terminus or somewhere in between) and/or theregulatory element of the NS1 gene. In one embodiment, an attenuatedinfluenza virus comprises a genome having a mutation in an influenzavirus NS1 gene resulting in a deletion consisting of 5, preferably 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 75, 80, 85, 90, 95, 99, 100,105, 110, 115, 120, 125, 126, 130, 135, 140, 145, 150, 155, 160, 165,170 or 175 amino acid residues from the C-terminus of NS1, or a deletionof between 5-170, 25-170, 50-170, 100-170, 100-160, or 105-160 aminoacid residues from the C-terminus. In another embodiment, an attenuatedinfluenza virus comprises a genome having a mutation in an influenzavirus NS1 gene such that it encodes an NS1 protein of amino acidresidues 1-130, amino acid residues 1-126, amino acid residues 1-120,amino acid residues 1-115, amino acid residues 1-110, amino acidresidues 1-100, amino acid residues 1-99, amino acid residues 1-95,amino acid residues 1-85, amino acid residues 1-83, amino acid residues1-80, amino acid residues 1-75, amino acid residues 1-73, amino acidresidues 1-70, amino acid residues 1-65, or amino acid residues 1-60,wherein the N-terminus amino acid is number 1. For examples of NS1mutations and influenza viruses comprising a mutated NS1, see, e.g.,U.S. Pat. Nos. 6,468,544 and 6,669,943; and Li et al., 1999, J. Infect.Dis. 179:1132-1138, each of which is incorporated by reference herein inits entirety.

5.5 Non-Influenza Virus Vectors

In one aspect, provided herein are non-influenza viruses containing aninfluenza hemagglutinin stem domain polypeptide. In a specificembodiment, the influenza hemagglutinin stem domain polypeptide isincorporated into the virions of the non-influenza virus. Thenon-influenza viruses may be conjugated to moieties that target theviruses to particular cell types, such as immune cells. In someembodiments, the virions of the non-influenza virus have incorporatedinto them or express a heterologous polypeptide in addition to aninfluenza hemagglutinin stem domain polypeptide. The heterologouspolypeptide may be a polypeptide that has immunopotentiating activity,or that targets the non-influenza virus to a particular cell type, suchas an antibody that recognizes an antigen on a specific cell type or aligand that binds a specific receptor on a specific cell type. SeeSection 5.4 supra for examples of such heterologous polypeptides.

Non-influenza viruses containing an influenza hemagglutinin stem domainpolypeptide may be produced by supplying in trans the influenzahemagglutinin stem domain polypeptide during production of virions usingtechniques known to one skilled in the art. Alternatively, thereplication of a parental non-influenza virus comprising a genomeengineered to express an influenza hemagglutinin stem domain polypeptidein cells susceptible to infection with the virus wherein hemagglutininfunction is provided in trans will produce progeny viruses containingthe influenza hemagglutinin stem domain polypeptide.

Any virus type, subtype or strain including, but not limited to,naturally occurring strains, variants or mutants, mutagenized viruses,reassortants and/or genetically modified viruses may be used as anon-influenza virus vector. In a specific embodiment, the parentalnon-influenza virus is not a naturally occurring virus. In anotherspecific embodiment, the parental non-influenza virus is a geneticallyengineered virus. In certain embodiments, an enveloped virus ispreferred for the expression of a membrane bound influenza hemagglutininstem domain polypeptide described herein.

In an exemplary embodiment, the non-influenza virus vector is aNewcastle disease virus (NDV). In another embodiment, the non-influenzavirus vector is a vaccinia virus. In other exemplary, non-limiting,embodiments, the non-influenza virus vector is adenovirus,adeno-associated virus (AAV), hepatitis B virus, retrovirus (such as,e.g., a gammaretrovirus such as Mouse Stem Cell Virus (MSCV) genome orMurine Leukemia Virus (MLV), e.g., Moloney murine leukemia virus,oncoretrovirus, or lentivirus), an alphavirus (e.g., Venezuelan equineencephalitis virus), a rhabdovirus, such as vesicular stomatitis virusor papillomaviruses, poxvirus (such as, e.g., vaccinia virus, a MVA-T7vector, or fowlpox), metapneumovirus, measles virus, herpesvirus, suchas herpes simplex virus, or foamyvirus. See, e.g., Lawrie and Tumin,1993, Cur. Opin. Genet. Develop. 3, 102-109 (retroviral vectors); Bettet al., 1993, J. Virol. 67, 5911 (adenoviral vectors); Zhou et al.,1994, J. Exp. Med. 179, 1867 (adeno-associated virus vectors); Dubenskyet al., 1996, J. Virol. 70, 508-519 (viral vectors from the pox familyincluding vaccinia virus and the avian pox viruses and viral vectorsfrom the alpha virus genus such as those derived from Sindbis andSemliki Forest Viruses); U.S. Pat. No. 5,643,576 (Venezuelan equineencephalitis virus); WO 96/34625 (VSV); Ohe et al., 1995, Human GeneTherapy 6, 325-333; Woo et al., WO 94/12629; Xiao & Brandsma, 1996,Nucleic Acids. Res. 24, 2630-2622 (papillomaviruses); and Bukreyev andCollins, 2008, Curr Opin Mol Ther. 10:46-55 (NDV), each of which isincorporated by reference herein in its entirety.

In a specific embodiment, the non-influenza virus vector is NDV. Any NDVtype, subtype or strain may serve as the backbone that is engineered toexpress an influenza hemagglutinin stem domain polypeptide, including,but not limited to, naturally-occurring strains, variants or mutants,mutagenized viruses, reassortants and/or genetically engineered viruses.In a specific embodiment, the NDV that serves as the backbone forgenetic engineering is a naturally-occurring strain. In certainembodiments, the NDV that serves as the backbone for genetic engineeringis a lytic strain. In other embodiments, the NDV that serves as thebackbone for genetic engineering is a non-lytic strain. In certainembodiments, the NDV that serves as the backbone for genetic engineeringis lentogenic strain. In some embodiments, the NDV that serves as thebackbone for genetic engineering is a mesogenic strain. In otherembodiments, the NDV that serves as the backbone for genetic engineeringis a velogenic strain. Specific examples of NDV strains include, but arenot limited to, the 73-T strain, Ulster strain, MTH-68 strain, Italienstrain, Hickman strain, PV701 strain, Hitchner B1 strain, La Sotastrain, YG97 strain, MET95 strain, and F48E9 strain. In a specificembodiment, the NDV that serves as the backbone for genetic engineeringis the Hitchner B1 strain. In another specific embodiment, the NDV thatserves as the backbone for genetic engineering is the La Sota strain.

In one embodiment, the NDV used as the backbone for a non-influenzavirus vector is engineered to express a modified F protein in which thecleavage site of the F protein is replaced with one containing one ortwo extra arginine residues, allowing the mutant cleavage site to beactivated by ubiquitously expressed proteases of the furin family.Specific examples of NDVs that express such a modified F proteininclude, but are not limited to, rNDV/F2aa and rNDV/F3aa. For adescription of mutations introduced into a NDV F protein to produce amodified F protein with a mutated cleavage site, see, e.g., Park et al.(2006) “Engineered viral vaccine constructs with dual specificity: Avianinfluenza and Newcastle disease.” PNAS USA 103: 8203-2808, which isincorporated herein by reference in its entirety.

In one embodiment, the non-influenza virus vector is a poxvirus. Apoxvirus vector may be based on any member of the poxviridae, inparticular, a vaccinia virus or an avipox virus (e.g., such ascanarypox, fowlpox, etc.) that provides suitable sequences for vaccinevectors. In a specific embodiment, the poxviral vector is a vacciniavirus vector. Suitable vaccinia viruses include, but are not limited to,the Copenhagen (VC-2) strain (Goebel, et al., Virol 179: 247-266, 1990;Johnson, et al., Virol. 196: 381-401, 1993), modified Copenhagen strain(NYVAC) (U.S. Pat. No. 6,265,189), the WYETH strain and the modifiedAnkara (MVA) strain (Antoine, et al., Virol. 244: 365-396, 1998). Othersuitable poxviruses include fowlpox strains such as ALVAC and TROVACvectors that provide desirable properties and are highly attenuated(see, e.g., U.S. Pat. No. 6,265,189; Tartaglia et al., In AIDS ResearchReviews, Koff, et al., eds., Vol. 3, Marcel Dekker, N.Y., 1993; andTartaglia et al., 1990, Reviews in Immunology 10: 13-30, 1990).

Methods of engineering non-influenza viruses to express an influenzahemagglutinin stem domain polypeptide are well known in the art, as aremethods for attenuating, propagating, and isolating and purifying suchviruses. For such techniques with respect to NDV vectors, see, e.g.,International Publication No. WO 01/04333; U.S. Pat. Nos. 7,442,379,6,146,642, 6,649,372, 6,544,785 and 7,384,774; Swayne et al. (2003).Avian Dis. 47:1047-1050; and Swayne et al. (2001). J. Virol.11868-11873, each of which is incorporated by reference in its entirety.For such techniques with respect to poxviruses, see, e.g., Piccini, etal., Methods of Enzymology 153: 545-563, 1987; International PublicationNo. WO 96/11279; U.S. Pat. No. 4,769,330; U.S. Pat. No. 4,722,848; U.S.Pat. No. 4,769,330; U.S. Pat. No. 4,603,112; U.S. Pat. No. 5,110,587;U.S. Pat. No. 5,174,993; EP 83 286; EP 206 920; Mayr et al., Infection3: 6-14, 1975; and Sutter and Moss, Proc. Natl. Acad. Sci. USA 89:10847-10851, 1992. In certain embodiments, the non-influenza virus isattenuated.

Exemplary considerations for the selection of a non-influenza virusvector, particularly for use in compositions for administration to asubject, are safety, low toxicity, stability, cell type specificity, andimmunogenicity, particularly, antigenicity of the influenzahemagglutinin stem domain polypeptide expressed by the non-influenzavirus vector.

5.6 Viral-Like Particles and Virosomes

Influenza hemagglutinin stem domain polypeptides can be incorporatedinto viral-like particle (VLP) vectors. VLPs generally comprise a viralpolypeptide(s) typically derived from a structural protein(s) of avirus. In some embodiments, the VLPs are not capable of replicating. Incertain embodiments, the VLPs may lack the complete genome of a virus orcomprise a portion of the genome of a virus. In some embodiments, theVLPs are not capable of infecting a cell. In some embodiments, the VLPsexpress on their surface one or more of viral (e.g., virus surfaceglycoprotein) or non-viral (e.g., antibody or protein) targetingmoieties known to one skilled in the art or described herein. In someembodiments, the VLPs comprise an influenza hemagglutinin stem domainpolpeptide and a viral structural protein, such as HIV gag. In aspecific embodiment, the VLPs comprise an influenza hemagglutinin stemdomain polypeptide and an HIV gag polypeptide, such as described inExample 2 in Section 6.2 below.

Methods for producing and characterizing recombinantly produced VLPshave been described based on several viruses, including influenza virus(Bright et al. (2007) Vaccine. 25:3871), human papilloma virus type 1(Hagnesee et al. (1991) J. Virol. 67:315), human papilloma virus type 16(Kirnbauer et al. Proc. Natl. Acad. Sci. (1992) 89:12180), HIV-1 (Hafferet al., (1990) J. Virol. 64:2653), and hepatitis A (Winokur (1991)65:5029), each of which is incorporated herein in its entirety. Methodsfor expressing VLPs that contain NDV proteins are provided by Pantua etal. (2006) J. Virol. 80:11062-11073, and in United States patentapplication Publication No. 20090068221, published Mar. 12, 2009, eachof which is incorporated in its entirety herein.

In a specific embodiment, an influenza hemagglutinin stem domainpolypeptide may be incorporated into a virosome. A virosome containingan influenza hemagglutinin stem domain polypeptide may be produced usingtechniques known to those skilled in the art. For example, a virosomemay be produced by disrupting a purified virus, extracting the genome,and reassembling particles with the viral proteins (e.g., an influenzahemagglutinin stem domain polypeptide) and lipids to form lipidparticles containing viral proteins.

5.7 Bacterial Vectors

In a specific embodiment, bacteria may be engineered to express aninfluenza hemagglutinin stem domain polypeptide described herein.Suitable bacteria for expression of an influenza virus hemagglutininstem domain include, but are not limited to, Listeria, Salmonella,Shigella sp., Mycobacterium tuberculosis, E. coli, Neisseriameningitides, Brucella abortus, Brucella melitensis, Borreliaburgdorferi, and Francisella tularensis. In a specific embodiment, thebacteria engineered to express an influenza hemagglutinin stem domainpolypeptide are attenuated. Techniques for the production of bacteriaengineered to express a heterologous polypeptide are known in the artand can be applied to the expression of an influenza hemagglutinin stemdomain polypeptide. See, e.g., United States Patent ApplicationPublication No. 20080248066, published Oct. 9, 2008, and United StatesPatent Application Publication No. 20070207171, published Sep. 6, 2007,each of which are incorporated by reference herein in their entirety.

5.8 Plant and Algae Vectors

In certain embodiments, plants (e.g., plants of the genus Nicotiana) maybe engineered to express an influenza hemagglutinin stem domainpolypeptide described herein. In specific embodiments, plants areengineered to express an influenza hemagglutinin stem domain polypeptidedescribed herein via an agroinfiltration procedure using methods knownin the art. For example, nucleic acids encoding a gene of interest,e.g., a gene encoding influenza hemagglutinin stem domain polypeptidedescribed herein, are introduced into a strain of Agrobacterium.Subsequently the strain is grown in a liquid culture and the resultingbacteria are washed and suspended into a buffer solution. The plants arethen exposed (e.g., via injection or submersion) to the Agrobacteriumthat comprises the nucleic acids encoding an influenza hemagglutininstem domain polypeptide described herein such that the Agrobacteriumtransforms the gene of interest to a portion of the plant cells. Theinfluenza hemagglutinin stem domain polypeptide is then transientlyexpressed by the plant and can isolated using methods known in the artand described herein. (For specific examples see Shoji et al., 2008,Vaccine, 26(23):2930-2934; and D'Aoust et al., 2008, J. PlantBiotechnology, 6(9):930-940). In a specific embodiment, the plant is atobacco plant (i.e., Nicotiana tabacum). In another specific embodiment,the plant is a relative of the tobacco plant (e.g., Nicotianabenthamiana).

In other embodiments, algae (e.g., Chlamydomonas reinhardtii) may beengineered to express an influenza hemagglutinin stem domain polypeptidedescribed herein (see, e.g., Rasala et al., 2010, Plant BiotechnologyJournal (Published online Mar. 7, 2010)).

5.9 Generation of Antibodies Against Influenza Hemagglutinin Stem DomainPolypeptide

The influenza hemagglutinin stem domain polypeptides, nucleic acidsencoding such polypeptides, or vectors comprising such nucleic acids orpolypeptides described herein may be used to elicit neutralizingantibodies against influenza, for example, against the stalk region ofinfluenza virus hemagglutinin polypeptide. In a specific embodiment, theinfluenza hemagglutinin stem domain polypeptides, nucleic acids encodingsuch polypeptides, or vectors comprising such nucleic acids orpolypeptides described herein may be administered to a non-human subject(e.g., a mouse, rabbit, rat, guinea pig, etc.) to induce an immuneresponse that includes the production of antibodies which may beisolated using techniques known to one of skill in the art (e.g.,immunoaffinity chromatography, centrifugation, precipitation, etc.).

Alternatively, influenza hemagglutinin stem domain polypeptidesdescribed herein may be used to screen for antibodies from antibodylibraries. For example, an isolated influenza hemagglutinin stem domainpolypeptide may be immobilized to a solid support (e.g., a silica gel, aresin, a derivatized plastic film, a glass bead, cotton, a plastic bead,a polystyrene bead, an alumina gel, or a polysaccharide, a magneticbead), and screened for binding to antibodies. As an alternative, theantibodies may be immobilized to a solid support and screened forbinding to the isolated influenza hemagglutinin stem domain polypeptide.Any screening assay, such as a panning assay, ELISA, surface plasmonresonance, or other antibody screening assay known in the art may beused to screen for antibodies that bind to the influenza hemagglutininstem domain. The antibody library screened may be a commerciallyavailable antibody library, an in vitro generated library, or a libraryobtained by identifying and cloning or isolating antibodies from anindividual infected with influenza. In particular embodiments, theantibody library is generated from a survivor of an influenza virusoutbreak. Antibody libraries may be generated in accordance with methodsknown in the art. In a particular embodiment, the antibody library isgenerated by cloning the antibodies and using them in phage displaylibraries or a phagemid display library.

Antibodies identified in the methods described herein may be tested forneutralizing activity and lack of autoreactivity using the biologicalassays known in the art or described herein. In one embodiment, anantibody isolated from a non-human animal or an antibody libraryneutralizes a hemagglutinin polypeptide from more than one influenzasubtype. In some embodiments, an antibody elicited or identified usingan influenza hemagglutinin stem domain polypeptide, a nucleic acidencoding such a polypeptide, or a vector encoding such a nucleic acid orpolypeptide neutralizes an influenza H3 virus. In some embodiments, anantibody elicited or identified using an influenza hemagglutinin stemdomain polypeptide, a nucleic acid encoding such a polypeptide, or avector comprising such a nucleic acid or polypeptide neutralizes 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 or more subtypes orstrains of influenza virus. In one embodiment, the neutralizing antibodyneutralizes one or more influenza A viruses and one or more influenza Bviruses. In particular embodiments, the neutralizing antibody is not, ordoes not bind the same epitope as CR6261, CR6325, CR6329, CR6307,CR6323, 2A, D7, D8, F10, G17, H40, A66, D80, E88, E90, H98, C179(produced by hybridoma FERM BP-4517; clones sold by Takara Bio, Inc.(Otsu, Shiga, Japan)), AI3C (produced by hybridoma FERM BP-4516) or anyother antibody described in Ekiert D C et al. (2009) AntibodyRecognition of a Highly Conserved Influenza Virus Epitope. Science(published in Science Express Feb. 26, 2009); Kashyap et al. (2008)Combinatorial antibody libraries from survivors of the Turkish H5N1avian influenza outbreak reveal virus neutralization strategies. ProcNatl Acad Sci USA 105: 5986-5991; Sui et al. (2009) Structural andfunctional bases for broad-spectrum neutralization of avian and humaninfluenza A viruses. Nat Struct Mol Biol 16: 265-273; U.S. Pat. Nos.5,589,174, 5,631,350, 6,337,070, and 6,720,409; InternationalApplication No. PCT/US2007/068983 published as International PublicationNo. WO 2007/134237; International Application No. PCT/US2008/075998published as International Publication No. WO 2009/036157; InternationalApplication No. PCT/EP2007/059356 published as International PublicationNo. WO 2008/028946; and International Application No. PCT/US2008/085876published as International Publication No. WO 2009/079259. In otherembodiments, the neutralizing antibody is not an antibody described inWang et al. (2010) “Broadly Protective Monoclonal Antibodies against H3Influenza Viruses following Sequential Immunization with DifferentHemagglutinins,” PLOS Pathogens 6(2):1-9. In particular embodiments, theneutralizing antibody does not use the Ig VH1-69 segment. In someembodiments, the interaction of the neutralizing antibody with theantigen is not mediated exclusively by the heavy chain.

Antibodies identified or elicited using an influenza hemagglutinin stemdomain polypeptide, a nucleic acid encoding such a polypeptide, or avector comprising such a nucleic acid or polypeptide includeimmunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules, i.e., molecules that contain an antigenbinding site that specifically binds to a hemagglutinin polypeptide. Theimmunoglobulin molecules may be of any type (e.g., IgG, IgE, IgM, IgD,IgA and IgY), class (e.g., IgG₁, IgG₂, IgG₃, IgA₁ and IgA₂) or subclassof immunoglobulin molecule. Antibodies include, but are not limited to,monoclonal antibodies, multispecific antibodies, human antibodies,humanized antibodies, chimeric antibodies, single-chain Fvs (scFv),single chain antibodies, Fab fragments, F(ab′) fragments,disulfide-linked Fvs (sdFv), and anti-idiotypic (anti-Id) antibodies(including, e.g., anti-Id antibodies to antibodies elicited oridentified using a method described herein), and epitope-bindingfragments of any of the above.

Antibodies elicited or identified using an influenza hemagglutinin stemdomain polypeptide, nucleic acids encoding such a polypeptide or avector comprising such a nucleic acid or polypeptide may be used indiagnostic immunoassays, passive immunotherapy, and generation ofantiidiotypic antibodies. The antibodies before being used in passiveimmunotherapy may be modified, e.g., the antibodies may be chimerized orhumanized. See, e.g., U.S. Pat. Nos. 4,444,887 and 4,716,111; andInternational Publication Nos. WO 98/46645, WO 98/50433, WO 98/24893, WO98/16654, WO 96/34096, WO 96/33735, and WO 91/10741, each of which isincorporated herein by reference in its entirety, for reviews on thegeneration of chimeric and humanized antibodies. In addition, theability of the antibodies to neutralize hemagglutinin polypeptides andthe specificity of the antibodies for the polypeptides may be testedprior to using the antibodies in passive immunotherapy. See Section 5.11infra for a discussion regarding use of neutralizing antibodies for theprevention or treatment of disease caused by influenza virus infection.

Antibodies elicited or identified using an influenza hemagglutinin stemdomain polypeptide, a nucleic acid encoding such a polypeptide, or avector comprising such a nucleic acid or polypeptide may be used tomonitor the efficacy of a therapy and/or disease progression. Anyimmunoassay system known in the art may be used for this purposeincluding, but not limited to, competitive and noncompetitive assaysystems using techniques such as radioimmunoassays, ELISA (enzyme linkedimmunosorbent assays), “sandwich” immunoassays, precipitin reactions,gel diffusion precipitin reactions, immunodiffusion assays,agglutination assays, complement fixation assays, immunoradiometricassays, fluorescent immunoassays, protein A immunoassays andimmunoelectrophoresis assays, to name but a few.

Antibodies elicited or identified using an influenza hemagglutinin stemdomain polypeptide, a nucleic acid encoding such a polypeptide, or avector comprising such a nucleic acid or polypeptide may be used in theproduction of antiidiotypic antibody. The antiidiotypic antibody canthen in turn be used for immunization, in order to produce asubpopulation of antibodies that bind a particular antigen of influenza,e.g., a neutralizing epitope of a hemagglutinin polypeptide (Jerne,1974, Ann. Immunol. (Paris) 125c:373; Jerne et al., 1982, EMBO J. 1:234,incorporated herein by reference in its entirety).

5.10 Stimulation of Cells with Influenza Hemagglutinin Stem DomainPeptide

In another aspect, provided herein are methods for stimulating cells exvivo with an influenza hemagglutinin stem domain polypeptide describedherein. Such cells, e.g., dendritic cells, may be used in vitro togenerate antibodies against the influenza hemagglutinin stem domainpolypeptide or may themselves be administered to a subject by, e.g., anadoptive transfer technique known in the art. See, e.g., United Statespatent application Publication No. 20080019998, published Jan. 24, 2008,which is incorporated herein by reference in its entirety, for adescription of adoptive transfer techniques. In certain embodiments,when cells that have been stimulated ex vivo with an influenzahemagglutinin stem domain polypeptide described herein are administeredto a subject, the cells are not mammalian cells (e.g., CB-1 cells).

In one non-limiting example, a vector, e.g., an influenza virus vector,engineered to express an influenza hemagglutinin stem domain polypeptidedescribed herein can be used to generate dendritic cells (DCs) thatexpress the influenza hemagglutinin stem domain polypeptide and displayimmunostimulatory properties directed against an influenza virushemagglutinin polypeptide. Such DCs may be used to expand memory T cellsand are potent stimulators of T cells, including influenza hemagglutininstem domain polypeptide-specific cytotoxic T lymphocyte clones. SeeStrobel et al., 2000, Human Gene Therapy 11:2207-2218, which isincorporated herein by reference in its entirety.

An influenza hemagglutinin stem domain polypeptide described herein maybe delivered to a target cell in any way that allows the polypeptide tocontact the target cell, e.g., a DC, and deliver the polypeptide to thetarget cell. In certain embodiments, the influenza hemagglutinin stemdomain polypeptide is delivered to a subject, as described herein. Insome such embodiments, cells contacted with the polypeptide may beisolated and propagated.

In certain embodiments, an influenza hemagglutinin stem domainpolypeptide is delivered to a target cell in vitro. Techniques known toone of skill in the art may be used to deliver the polypeptide to targetcells. For example, target cells may be contacted with the polypeptidein a tissue culture plate, tube or other container. The polypeptide maybe suspended in media and added to the wells of a culture plate, tube orother container. The media containing the polypeptide may be added priorto plating of the cells or after the cells have been plated. The targetcells are preferably incubated with the polypeptide for a sufficientamount of time to allow the polypeptide to contact the cells. In certainembodiments, the cells are incubated with the polypeptide for about 1hour or more, about 5 hours or more, about 10 hours or more, about 12hours or more, about 16 hours or more, about 24, hours or more, about 48hours or more, about 1 hour to about 12 hours, about 3 hours to about 6hours, about 6 hours to about 12 hours, about 12 hours to about 24hours, or about 24 hours to about 48 hours. In certain embodiments,wherein the influenza hemagglutinin stem domain polypeptide is in avirus, the contacting of the target cells comprises infecting the cellswith the virus.

The target cells may be from any species, including, e.g., humans, mice,rats, rabbits and guinea pigs. In some embodiments, target cells are DCsobtained from a healthy subject or a subject in need of treatment. Incertain embodiments, target cells are DCs obtained from a subject inwhom it is desired to stimulate an immune response to the polypeptide.Methods of obtaining cells from a subject are well known in the art.

5.11 Compositions

The nucleic acids, vectors, polypeptides, bacteria, antibodies, or cellsdescribed herein (sometimes referred to herein as “active compounds”)may be incorporated into compositions. In a specific embodiment, thecompositions are pharmaceutical compositions, such as immunogeniccompositions (e.g., vaccine formulations). The pharmaceuticalcompositions provided herein can be in any form that allows for thecomposition to be administered to a subject. In a specific embodiment,the pharmaceutical compositions are suitable for veterinary and/or humanadministration. The compositions may be used in methods of preventing ortreating an influenza virus disease.

In one embodiment, a pharmaceutical composition comprises an influenzahemagglutinin stem domain polypeptide, in an admixture with apharmaceutically acceptable carrier. In another embodiment, apharmaceutical composition comprises a nucleic acid encoding aninfluenza hemagglutinin stem domain polypeptide described herein, in anadmixture with a pharmaceutically acceptable carrier. In anotherembodiment, a pharmaceutical composition comprises an expression vectorcomprising a nucleic acid encoding an influenza hemagglutinin stemdomain polypeptide, in an admixture with a pharmaceutically acceptablecarrier. In another embodiment, a pharmaceutical composition comprisesan influenza virus or non-influenza virus containing an influenzahemagglutinin stem domain polypeptide, in an admixture with apharmaceutically acceptable carrier. In another embodiment, apharmaceutical composition comprises an influenza virus or non-influenzavirus having a genome engineered to express an influenza hemagglutininstem domain polypeptide, in admixture with a pharmaceutically acceptablecarrier. In another embodiment, a pharmaceutical composition comprises aviral-like particle or virosome containing an influenza hemagglutininstem domain polypeptide, in an admixture with a pharmaceuticallyacceptable carrier. In another embodiment, a pharmaceutical compositioncomprises a bacteria expressing or engineered to express an influenzahemagglutinin stem domain polypeptide, in an admixture with apharmaceutically acceptable carrier. In another embodiment, apharmaceutical composition comprises cells stimulated with an influenzahemagglutinin stem domain polypeptide, in an admixture with apharmaceutically acceptable carrier.

In some embodiments, a pharmaceutical composition may comprise one ormore other therapies in addition to an active compound.

As used herein, the term “pharmaceutically acceptable” means approved bya regulatory agency of the Federal or a state government or listed inthe U.S. Pharmacopeia or other generally recognized pharmacopeiae foruse in animals, and more particularly in humans. The term “carrier”refers to a diluent, adjuvant, excipient, or vehicle with which thepharmaceutical composition is administered. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable excipients includestarch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,silica gel, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, propylene, glycol, water, ethanoland the like. Examples of suitable pharmaceutical carriers are describedin “Remington's Pharmaceutical Sciences” by E. W. Martin. Theformulation should suit the mode of administration.

In a specific embodiment, pharmaceutical compositions are formulated tobe suitable for the intended route of administration to a subject. Forexample, the pharmaceutical composition may be formulated to be suitablefor parenteral, oral, intradermal, transdermal, colorectal,intraperitoneal, and rectal administration. In a specific embodiment,the pharmaceutical composition may be formulated for intravenous, oral,intraperitoneal, intranasal, intratracheal, subcutaneous, intramuscular,topical, intradermal, transdermal or pulmonary administration.

In certain embodiments, biodegradable polymers, such as ethylene vinylacetate, polyanhydrides, polyethylene glycol (PEGylation), polymethylmethacrylate polymers, polylactides, poly(lactide-co-glycolides),polyglycolic acid, collagen, polyorthoesters, and polylactic acid, maybe used as carriers. In some embodiments, the active compounds areprepared with carriers that increase the protection of the compoundagainst rapid elimination from the body, such as a controlled releaseformulation, including implants and microencapsulated delivery systems.Methods for preparation of such formulations will be apparent to thoseskilled in the art. Liposomes or micelles can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811. In certain embodiments, the pharmaceuticalcompositions comprise one or more adjuvants.

In specific embodiments, immunogenic compositions described herein aremonovalent formulations. In other embodiments, immunogenic compositionsdescribed herein are multivalent formulations. In one example, amultivalent formulation comprises one or more vectors expressing aninfluenza hemagglutinin stem domain polypeptide derived from aninfluenza A virus hemagglutinin polypeptide and one or more vectorsexpressing an influenza hemagglutinin stem domain polypeptide derivedfrom an influenza B virus hemagglutinin polypeptide. In another example,a multivalent formulation comprises a vector expressing an influenzahemagglutinin stem domain polypeptide derived from an influenza A virusH3 antigen and a vector expressing an influenza hemagglutinin stemdomain polypeptide derived from an influenza A virus H1 antigen. Inanother example, a multivalent formulation comprises a vector expressingan influenza hemagglutinin stem domain polypeptide derived from aninfluenza A virus H3 antigen, a vector expressing an influenzahemagglutinin stem domain polypeptide derived from an influenza A virusH1 antigen, and a vector expressing an influenza hemagglutinin stemdomain polypeptide derived from an influenza B virus HA antigen. Incertain embodiments, a multivalent formulation may comprise one or moredifferent influenza hemagglutinin stem domain polypeptides expressedusing a single vector.

In certain embodiments, the pharmaceutical compositions described hereinadditionally comprise a preservative, e.g., the mercury derivativethimerosal. In a specific embodiment, the pharmaceutical compositionsdescribed herein comprises 0.001% to 0.01% thimerosal. In otherembodiments, the pharmaceutical compositions described herein do notcomprise a preservative. In a specific embodiment, thimerosal is usedduring the manufacture of a pharmaceutical composition described hereinand the thimerosal is removed via purification steps followingproduction of the pharmaceutical composition, i.e., the pharmaceuticalcomposition contains trace amounts of thimerosal (<0.3 μg of mercury perdose after purification; such pharmaceutical compositions are consideredthimerosal-free products).

In certain embodiments, the pharmaceutical compositions described hereinadditionally comprise egg protein (e.g., ovalbumin or other eggproteins). The amount of egg protein in the pharmaceutical compositionsdescribed herein may range from about 0.0005 to about 1.2. μg of eggprotein to 1 ml of pharmaceutical composition. In other embodiments, thepharmaceutical compositions described herein do not comprise eggprotein.

In certain embodiments, the pharmaceutical compositions described hereinadditionally comprise one or more antimicrobial agents (e.g.,antibiotics) including, but not limited to gentamicin, neomycin,polymyxin (e.g., polymyxin B), and kanamycin, streptomycin. In otherembodiments, the pharmaceutical compositions described herein do notcomprise any antibiotics.

In certain embodiments, the pharmaceutical compositions described hereinadditionally comprise one or more components used to inactivate a virus,e.g., formalin or formaldehyde or a detergent such as sodiumdeoxycholate, octoxynol 9 (Triton X-100), and octoxynol 10. In otherembodiments, the pharmaceutical compositions described herein do notcomprise any components used to inactivate a virus.

In certain embodiments, the pharmaceutical compositions described hereinadditionally comprise gelatin. In other embodiments, the pharmaceuticalcompositions described herein do not comprise gelatin.

In certain embodiments, the pharmaceutical compositions described hereinadditionally comprise one or more buffers, e.g., phosphate buffer andsucrose phosphate glutamate buffer. In other embodiments, thepharmaceutical compositions described herein do not comprise buffers.

In certain embodiments, the pharmaceutical compositions described hereinadditionally comprise one or more salts, e.g., sodium chloride, calciumchloride, sodium phosphate, monosodium glutamate, and aluminum salts(e.g., aluminum hydroxide, aluminum phosphate, alum (potassium aluminumsulfate), or a mixture of such aluminum salts). In other embodiments,the pharmaceutical compositions described herein do not comprise salts.

In specific embodiments, the the pharmaceutical compositions describedherein are low-additive influenza virus vaccines, i.e., thepharmaceutical compositions do not comprise one or more additivescommonly found in influenza virus vaccines. Low-additive influenzavaccines have been described (see, e.g., International Application No.PCT/IB2008/002238 published as International Publication No. WO09/001217 which is herein incorporated by reference in its entirety).

The pharmaceutical compositions described herein can be included in acontainer, pack, or dispenser together with instructions foradministration.

The pharmaceutical compositions described herein can be stored beforeuse, e.g., the pharmaceutical compositions can be stored frozen (e.g.,at about −20° C. or at about −70° C.); stored in refrigerated conditions(e.g., at about 4° C.); or stored at room temperature (see InternationalApplication No. PCT/IB2007/001149 published as International PublicationNo. WO 07/110776, which is herein incorporated by reference in itsentirety, for methods of storing compositions comprising influenzavaccines without refrigeration).

In certain embodiments, when the active compound in a pharmaceuticalcomposition described herein is a cell engineered to express aninfluenza hemagglutinin stem domain polypeptide, the cells in thepharmaceutical composition are not mammalian cells (e.g., CB-1 cells).

5.11.1 Subunit Vaccines

In a specific embodiment, provided herein are subunit vaccinescomprising an influenza hemagglutinin stem domain polypeptide describedherein. In some embodiments, a subunit vaccine comprises an influenzahemagglutinin stem domain polypeptide and one or more surfaceglycoproteins (e.g., influenza virus neuraminidase), other targetingmoieties or adjuvants. In specific embodiments, a subunit vaccinecomprises a single influenza hemagglutinin stem domain polypeptide. Inother embodiments, a subunit vaccine comprises two, three, four or moreinfluenza hemagglutinin stem domain polypeptides. In specificembodiments, the influenza hemagglutinin stem domain polypeptide(s) usedin a subunit vaccine is not membrane-bound, i.e., it is soluble.

In certain embodiments, provided herein are subunit vaccines comprisingabout 10 μg to about 60 μg of one or more influenza hemagglutinin stemdomain polypeptides described herein, about 0.001% to 0.01% thimerosal,about 0.1 μg to about 1.0 μg chicken egg protein, about 1.0 μg to about5.0 μg polymyxin, about 1.0 μg to about 5.0 neomycin, about 0.1 μg toabout 0.5 μg betapropiolactone, and about 0.001 to about 0.05% w/v ofnonylphenol ethoxylate per dose.

In a specific embodiment, a subunit vaccine provided herein comprises orconsists of a 0.5 ml dose that comprises 45 μg of influenzahemagglutinin stem domain polypeptide(s) provided herein, ≦1.0 μg ofmercury (from thimerosal), ≦1.0 μg chicken egg protein (i.e.,ovalbumin), ≦3.75 μg polymyxin, and ≦2.5 μg neomycin. In someembodiments, a subunit vaccine provided herein additionally comprises orconsists of not more than 0.5 μg betapropiolactone, and not more than0.015% w/v of nonylphenol ethoxylate per dose. In some embodiments, the0.5 ml dose subunit vaccine is packaged in a pre-filled syringe.

In a specific embodiment, a subunit vaccine provided herein consists ofa 5.0 ml multidose vial (0.5 ml per dose) that comprises 45 μg ofinfluenza hemagglutinin stem domain polypeptide(s) provided herein, 25.0μg of mercury (from thimerosal), ≦1.0 chicken egg protein (i.e.,ovalbumin), ≦3.75 μg polymyxin, and ≦2.5 μg neomycin. In someembodiments, a subunit vaccine provided herein additionally comprises orconsists of not more than 0.5 μg betapropiolactone, and not more than0.015% w/v of nonylphenol ethoxylate per dose.

In a specific embodiment, the subunit vaccine is prepared usinginfluenza virus that was propagated in embryonated chicken eggs (i.e.,the components of the subunit vaccine (e.g., a hemagglutinin stem domainpolypeptide) are isolated from virus that was propagated in embryonatedchicken eggs). In another specific embodiment, the subunit vaccine isprepared using influenza virus that was not propagated in embryonatedchicken eggs (i.e., the components of the subunit vaccine (e.g., ahemagglutinin stem domain polypeptide) are isolated from virus that wasnot propagated in embryonated chicken eggs). In another specificembodiment, the subunit vaccine is prepared using influenza virus thatwas propagated in mammalian cells, e.g., immortalized human cells (see,e.g., International Application No. PCT/EP2006/067566 published asInternational Publication No. WO 07/045674 which is herein incorporatedby reference in its entirety) or canine kidney cells such as MDCK cells(see, e.g., International Application No. PCT/IB2007/003536 published asInternational Publication No. WO 08/032219 which is herein incorporatedby reference in its entirety) (i.e., the components of the subunitvaccine (e.g., a hemagglutinin stem domain polypeptide) are isolatedfrom virus that was propagated in mammalian cells). In another specificembodiment, the hemagglutinin stem domain polypeptide(s) in a subunitvaccine are prepared using an expression vector, e.g., a viral vector,plant vector or a bacterial vector (i.e., the hemagglutinin stem domainpolypeptide(s) in the subunit vaccine are obtained/isolated from anexpression vector).

5.11.2 Live Virus Vaccines

In one embodiment, provided herein are immunogenic compositions (e.g.,vaccines) comprising live virus containing an influenza hemagglutininstem domain polypeptide. In another embodiment, provided herein areimmunogenic compositions (e.g., vaccines) comprising live virus that isengineered to encode an influenza hemagglutinin stem domain polypeptide,which is expressed by progeny virus produced in the subjectsadministered the compositions. In specific embodiments, the influenzahemagglutinin stem domain polypeptide is membrane-bound. In otherspecific embodiments, the influenza virus hemagglutinin stem domainpolypeptide is not membrane-bound, i.e., soluble. In particularembodiments, the live virus is an influenza virus, such as described inSection 5.4, supra. In other embodiments, the live virus is anon-influenza virus, such as described in Section 5.5, supra. In someembodiments, the live virus is attenuated. In some embodiments, animmunogenic composition comprises two, three, four or more live virusescontaining or engineered to express two, three, four or more differentinfluenza hemagglutinin stem domain polypeptides.

In certain embodiments, provided herein are immunogenic compositions(e.g., vaccines) comprising about 10⁵ to about 10¹⁰ fluorescent focusunits (FFU) of live attenuated influenza virus containing one or moreinfluenza hemagglutinin stem domain polypeptides described herein, about0.1 to about 0.5 mg monosodium glutamate, about 1.0 to about 5.0 mghydrolyzed procine gelatin, about 1.0 to about 5.0 mg arginine, about 10to about 15 mg sucrose, about 1.0 to about 5.0 mg dibasic potassiumphosphate, about 0.5 to about 2.0 mg monobasic potassium phosphate, andabout 0.001 to about 0.05 μg/ml gentamicin sulfate per dose. In someembodiments, the immunogenic compositions (e.g., vaccines) are packagedas pre-filled sprayers containing single 0.2 ml doses.

In a specific embodiment, provided herein are immunogenic compositions(e.g., vaccines) comprising 10^(6.5) to 10^(7.5) FFU of live attenuatedinfluenza virus containing one or more influenza hemagglutinin stemdomain polypeptides described herein, 0.188 mg monosodium glutamate, 2.0mg hydrolyzed procine gelatin, 2.42 mg arginine, 13.68 mg sucrose, 2.26mg dibasic potassium phosphate, 0.96 mg monobasic potassium phosphate,and <0.015 μg/ml gentamicin sulfate per dose. In some embodiments, theimmunogenic compositions (e.g., vaccines) are packaged as pre-filledsprayers containing single 0.2 ml doses.

In a specific embodiment, the live virus that contains an influenzahemagglutinin stem domain polypeptide is propagated in embryonatedchicken eggs before its use in an immunogenic composition describedherein. In another specific embodiment, the live virus that contains aninfluenza hemagglutinin stem domain polypeptide is not propagated inembryonated chicken eggs before its use in an immunogenic compositiondescribed herein. In another specific embodiment, the live virus thatcontains an influenza hemagglutinin stem domain polypeptide ispropagated in mammalian cells, e.g., immortalized human cells (see,e.g., International Application No. PCT/EP2006/067566 published asInternational Publication No. WO 07/045674 which is herein incorporatedby reference in its entirety) or canine kidney cells such as MDCK cells(see, e.g., International Application No. PCT/IB2007/003536 published asInternational Publication No. WO 08/032219 which is herein incorporatedby reference in its entirety) before its use in an immunogeniccomposition described herein.

An immunogenic composition comprising a live virus for administration toa subject may be preferred because multiplication of the virus in thesubject may lead to a prolonged stimulus of similar kind and magnitudeto that occurring in natural infections, and therefore, confersubstantial, long lasting immunity.

5.11.3 Inactivated Virus Vaccines

In one embodiment, provided herein are immunogenic compositions (e.g.,vaccines) comprising an inactivated virus containing an influenzahemagglutinin stem domain polypeptide. In specific embodiments, theinfluenza hemagglutinin stem domain polypeptide is membrane-bound. Inparticular embodiments, the inactivated virus is an influenza virus,such as described in Section 5.4, supra. In other embodiments, theinactivated virus is a non-influenza virus, such as described in Section5.5, supra. In some embodiments, an immunogenic composition comprisestwo, three, four or more inactivated viruses containing two, three, fouror more different influenza hemagglutinin stem domain polypeptides. Incertain embodiments, the inactivated virus immunogenic compositionscomprise one or more adjuvants.

Techniques known to one of skill in the art may be used to inactivateviruses containing an influenza hemagglutinin stem domain polypeptide.Common methods use formalin, heat, or detergent for inactivation. See,e.g., U.S. Pat. No. 6,635,246, which is herein incorporated by referencein its entirety. Other methods include those described in U.S. Pat. Nos.5,891,705; 5,106,619 and 4,693,981, which are incorporated herein byreference in their entireties.

In certain embodiments, provided herein are immunogenic compositions(e.g., vaccines) comprising inactivated influenza virus such that eachdose of the immunogenic composition comprises about 15 to about 60 μg ofinfluenza hemagglutinin stem domain polypeptide described herein, about1.0 to about 5.0 mg sodium chloride, about 20 to about 100 μg monobasicsodium phosphate, about 100 to about 500 μg dibasic sodium phosphate,about 5 to about 30 μg monobasic potassium phosphate, about 5 to about30 μg potassium chloride, and about 0.5 to about 3.0 μg calciumchloride. In some embodiments, the immunogenic compositions (e.g.,vaccines) are packaged as single 0.25 ml or single 0.5 ml doses. Inother embodiments, the immunogenic compositions (e.g., vaccines) arepackaged as multi-dose formulations.

In certain embodiments, provided herein are immunogenic compositions(e.g., vaccines) comprising inactivated influenza virus such that eachdose of the immunogenic composition comprises about 15 to about 60 μg ofinfluenza hemagglutinin stem domain polypeptide described herein, about0.001% to 0.01% thimerosal, about 1.0 to about 5.0 mg sodium chloride,about 20 to about 100 μg monobasic sodium phosphate, about 100 to about500 μg dibasic sodium phosphate, about 5 to about 30 μg monobasicpotassium phosphate, about 5 to about 30 μg potassium chloride, andabout 0.5 to about 3.0 μg calcium chloride per dose. In someembodiments, the immunogenic compositions (e.g., vaccines) are packagedas single 0.25 ml or single 0.5 ml doses. In other embodiments, theimmunogenic compositions (e.g., vaccines) are packaged as multi-doseformulations.

In a specific embodiment, immunogenic compositions (e.g., vaccines)provided herein are packaged as single 0.25 ml doses and comprise 22.5μg of influenza hemagglutinin stem domain polypeptide described herein,2.05 mg sodium chloride, 40 μg monobasic sodium phosphate, 150 μgdibasic sodium phosphate, 10 μg monobasic potassium phosphate, 10 μgpotassium chloride, and 0.75 μg calcium chloride per dose.

In a specific embodiment, immunogenic compositions (e.g., vaccines)provided herein are packaged as single 0.5 ml doses and comprise 45 μgof influenza hemagglutinin stem domain polypeptide described herein, 4.1mg sodium chloride, 80 μg monobasic sodium phosphate, 300 μg dibasicsodium phosphate, 20 μg monobasic potassium phosphate, 20 μg potassiumchloride, and 1.5 μg calcium chloride per dose.

In a specific embodiment, immunogenic compositions (e.g., vaccines) arepackaged as multi-dose formulations comprising or consisting of 5.0 mlof vaccine (0.5 ml per dose) and comprise 24.5 μg of mercury (fromthimerosal), 45 μg of influenza hemagglutinin stem domain polypeptidedescribed herein, 4.1 mg sodium chloride, 80 monobasic sodium phosphate,300 μg dibasic sodium phosphate, 20 μg monobasic potassium phosphate, 20μg potassium chloride, and 1.5 μg calcium chloride per dose.

In a specific embodiment, the inactivated virus that contains aninfluenza hemagglutinin stem domain polypeptide was propagated inembryonated chicken eggs before its inactivation and subsequent use inan immunogenic composition described herein. In another specificembodiment, the inactivated virus that contains an influenzahemagglutinin stem domain polypeptide was not propagated in embryonatedchicken eggs before its inactivation and subsequent use in animmunogenic composition described herein. In another specificembodiment, the inactivated virus that contains an influenzahemagglutinin stem domain polypeptide was propagated in mammalian cells,e.g., immortalized human cells (see, e.g., International Application No.PCT/EP2006/067566 published as International Publication No. WO07/045674 which is herein incorporated by reference in its entirety) orcanine kidney cells such as MDCK cells (see, e.g., InternationalApplication No. PCT/IB2007/003536 published as International PublicationNo. WO 08/032219 which is herein incorporated by reference in itsentirety) before its inactivation and subsequent use in an immunogeniccomposition described herein.

5.11.4 Split Virus Vaccines

In one embodiment, an immunogenic composition comprising an influenzahemagglutinin stem domain polypeptide is a split virus vaccine. In someembodiments, split virus vaccine contains two, three, four or moredifferent influenza hemagglutinin stem domain polypeptides. In certainembodiments, the influenza hemagglutinin stem domain polypeptide is/wasmembrane-bound. In certain embodiments, the split virus vaccinescomprise one or more adjuvants.

Techniques for producing split virus vaccines are known to those skilledin the art. By way of non-limiting example, an influenza virus splitvaccine may be prepared using inactivated particles disrupted withdetergents. One example of a split virus vaccine that can be adapted foruse in accordance with the methods described herein is the Fluzone®,Influenza Virus Vaccine (Zonal Purified, Subvirion) for intramuscularuse, which is formulated as a sterile suspension prepared from influenzaviruses propagated in embryonated chicken eggs. The virus-containingfluids are harvested and inactivated with formaldehyde. Influenza virusis concentrated and purified in a linear sucrose density gradientsolution using a continuous flow centrifuge. The virus is thenchemically disrupted using a nonionic surfactant, octoxinol-9, (Triton®X-100—A registered trademark of Union Carbide, Co.) producing a “splitvirus.” The split virus is then further purified by chemical means andsuspended in sodium phosphate-buffered isotonic sodium chloridesolution.

In certain embodiments, provided herein are split virus vaccinescomprising about 10 μg to about 60 μg of one or more influenzahemagglutinin stem domain polypeptides described herein, about 0.01 toabout 1.0 mg octoxynol-10 (TRITON X-100®, about 0.5 to 0.5 mgα-tocopheryl hydrogen succinate, about 0.1 to 1.0 mg polysorbate 80(Tween 80), about 0.001 to about 0.003 μg hydrocortisone, about 0.05 toabout 0.3 gentamcin sulfate, about 0.5 to about 2.0 μg chicken eggprotein (ovalbumin), about 25 to 75 μg formaldehyde, and about 25 to 75μg sodium deoxycholate.

In a specific embodiment, a split virus vaccine provided hereincomprises or consists of a 0.5 ml dose that comprises 45 μg of influenzahemagglutinin stem domain polypeptide(s) provided herein, ≦0.085 mgoctoxynol-10 (TRITON X-100®, ≦0.1 mg α-tocopheryl hydrogen succinate,≦0.415 mg polysorbate 80 (Tween 80), ≦0.0016 hydrocortisone, ≦0.15 μggentamcin sulfate, ≦1.0 chicken egg protein (ovalbumin), ≦50 μgformaldehyde, and ≦50 μg sodium deoxycholate. In some embodiments, the0.5 ml dose subunit vaccine is packaged in a pre-filled syringe.

In a specific embodiment, the split virus vaccine is prepared usinginfluenza virus that was propagated in embryonated chicken eggs. Inanother specific embodiment, the split virus vaccine is prepared usinginfluenza virus that was not propagated in embryonated chicken eggs. Inanother specific embodiment, the split virus vaccine is prepared usinginfluenza virus that was propagated in mammalian cells, e.g.,immortalized human cells (see, e.g., PCT/EP2006/067566 published as WO07/045674 which is herein incorporated by reference in its entirety) orcanine kidney cells such as MDCK cells (see, e.g., PCT/IB2007/003536published as WO 08/032219 which is herein incorporated by reference inits entirety).

5.11.5 Adjuvants

In certain embodiments, the compositions described herein comprise, orare administered in combination with, an adjuvant. The adjuvant foradministration in combination with a composition described herein may beadministered before, concommitantly with, or after administration ofsaid composition. In some embodiments, the term “adjuvant” refers to acompound that when administered in conjunction with or as part of acomposition described herein augments, enhances and/or boosts the immuneresponse to an influenza hemagglutinin stem domain polypeptide, but whenthe compound is administered alone does not generate an immune responseto the polypeptide. In some embodiments, the adjuvant generates animmune response to the polypeptide and does not produce an allergy orother adverse reaction. Adjuvants can enhance an immune response byseveral mechanisms including, e.g., lymphocyte recruitment, stimulationof B and/or T cells, and stimulation of macrophages.

In certain embodiments, an adjuvant augments the intrinsic response tothe influenza hemagglutinin stem domain polypeptide without causingconformational changes in the polypeptide that affect the qualitativeform of the response. Specific examples of adjuvants include, but arenot limited to, aluminum salts (alum) (such as aluminum hydroxide,aluminum phosphate, and aluminum sulfate), 3 De-O-acylatedmonophosphoryl lipid A (MPL) (see GB 2220211), MF59 (Novartis), AS03(GlaxoSmithKline), A504 (GlaxoSmithKline), polysorbate 80 (Tween 80; ICLAmericas, Inc.), imidazopyridine compounds (see InternationalApplication No. PCT/US2007/064857, published as InternationalPublication No. WO2007/109812), imidazoquinoxaline compounds (seeInternational Application No. PCT/US2007/064858, published asInternational Publication No. WO2007/109813) and saponins, such as QS21(see Kensil et al., in Vaccine Design: The Subunit and Adjuvant Approach(eds. Powell & Newman, Plenum Press, NY, 1995); U.S. Pat. No.5,057,540). In some embodiments, the adjuvant is Freund's adjuvant(complete or incomplete). Other adjuvants are oil in water emulsions(such as squalene or peanut oil), optionally in combination with immunestimulants, such as monophosphoryl lipid A (see Stoute et al., N. Engl.J. Med. 336, 86-91 (1997)). Another adjuvant is CpG (Bioworld Today,Nov. 15, 1998). Such adjuvants can be used with or without otherspecific immunostimulating agents such as MPL or 3-DMP, QS21, polymericor monomeric amino acids such as polyglutamic acid or polylysine, orother immunopotentiating agents described in Section 5.4, supra. Itshould be understood that different formulations of influenzahemagglutinin stem domain polypeptide may comprise different adjuvantsor may comprise the same adjuvant.

5.12 Prophylactic and Therapeutic Uses

In one aspect, provided herein are methods for inducing an immuneresponse in a subject utilizing an active compound, i.e., an influenzahemagglutinin stem domain polypeptide described herein, a nucleic acidencoding such a polypeptide, a vector (e.g., a viral vector, or abacteria) containing or expressing such a polypeptide, or cellsstimulated with such a polypeptide. In a specific embodiment, a methodfor inducing an immune response to an influenza virus hemagglutininpolypeptide in a subject comprises administering to a subject in needthereof an effective amount of an influenza virus hemagglutinin stemdomain polypeptide or an immunogenic composition thereof. In anotherembodiment, a method for inducing an immune response to an influenzavirus hemagglutinin polypeptide in a subject comprises administering toa subject in need thereof an effective amount of a nucleic acid encodingan influenza hemagglutinin stem domain polypeptide or an immunogeniccomposition thereof. In another embodiment, a method for inducing animmune response to an influenza virus hemagglutinin polypeptide in asubject comprises administering to a subject in need thereof aneffective amount of a viral vector containing or expressing an influenzahemagglutinin stem domain polypeptide or an immunogenic compositionthereof. In yet another embodiment, a method for inducing an immuneresponse to an influenza virus hemagglutinin polypeptide in a subjectcomprises administering to a subject in need thereof an effective amountof cells stimulated with an influenza hemagglutinin stem domainpolypeptide or a pharmaceutical composition thereof. In certainembodiments, an influenza hemagglutinin stem domain polypeptide used inthe method is a purified influenza hemagglutinin stem domain polypeptidederived from a mammalian cell, a plant cell, or an insect cell.

In a specific embodiment, a method for inducing an immune response to aninfluenza virus hemagglutinin polypeptide in a subject comprisesadministering to a subject in need thereof a subunit vaccine describedherein. In another embodiment, a method for inducing an immune responseto an influenza virus hemagglutinin polypeptide in a subject comprisesadministering to a subject in need thereof a live virus vaccinedescribed herein. In particular embodiments, the live virus vaccinecomprises an attenuated virus. In another embodiment, a method forinducing an immune response to an influenza virus hemagglutininpolypeptide in a subject comprises administering to a subject in needthereof an inactivated virus vaccine described herein. In anotherembodiment, a method for inducing an immune response to an influenzavirus hemagglutinin polypeptide in a subject comprises administering toa subject in need thereof a split virus vaccine described herein. Inanother embodiment, a method for inducing an immune response to aninfluenza virus hemagglutinin polypeptide in a subject comprisesadministering to a subject in need thereof a viral-like particle vaccinedescribed herein. In another embodiment, a method for inducing an immuneresponse to an influenza hemagglutinin polypeptide comprisesadministering to a subject in need thereof a virosome described herein.In another embodiment, a method for inducing an immune response to aninfluenza hemagglutinin polypeptide comprises administering to a subjectin need thereof a bacteria expressing or engineered to express aninfluenza hemagglutinin stem domain polypeptide or a compositionthereof. In certain embodiments, an influenza hemagglutinin stem domainpolypeptide used in the method is a purified influenza hemagglutininstem domain polypeptide derived from a mammalian cell, a plant cell, oran insect cell.

In some embodiments, the immune response induced by an active compoundor a composition described herein is effective to prevent and/or treatan influenza virus infection caused by any subtype or strain ofinfluenza virus. In certain embodiments, the immune response induced byan active compound or a composition described herein is effective toprevent and/or treat an influenza virus infection caused by a subtype ofinfluenza virus that belongs to one HA group (e.g., Group 1, whichcomprises H1, H2, H5, H6, H8, H9, H11, H12, H13, and H16) and not theother HA group (e.g., Group 2, which comprises H3, H4, H7, H10, H14, andH15). For example, the immune response induced may be effective toprevent and/or treat an influenza virus infection caused by an influenzavirus that belongs to the HA group consisting of H11, H13, H16, H9, H8,H12, H6, H1, H5 and H2. Alternatively, the immune response induced maybe effective to prevent and/or treat an influenza virus infection causedby an influenza virus that belongs to the HA group consisting of H3, H4,H14, H10, H15 and H7. In some embodiments, the immune response inducedby an active compound or a composition described herein is effective toprevent and/or treat an influenza virus infection caused by one, two,three, four or five subtypes of influenza virus. In certain embodiments,the immune response induced by an active compound or a compositiondescribed herein is effective to prevent and/or treat an influenza virusinfection caused by six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen or fifteen subtypes of influenza virus. In someembodiments, the immune response induced by an active compound or acomposition described herein is effective to prevent and/or treat aninfluenza virus infection caused by one or more variants within the samesubtype of influenza virus.

In some embodiments, the immune response induced by an active compoundor a composition described herein is effective to prevent and/or treatan influenza virus infection caused by both H1N1 and H2N2 subtypes. Inother embodiments, the immune response induced by an active compound ora composition described herein is not effective to prevent and/or treatan influenza virus infection caused by both H1N1 and H2N2 subtypes. Insome embodiments, the immune response induced by an active compound or acomposition described herein is effective to prevent and/or treat aninfluenza virus infection caused by H1N1, H2N2, and H3N2 subtypes. Insome embodiments, the immune response induced by an active compound or acomposition described herein is effective to prevent and/or treat aninfluenza virus infection caused by H3N2 subtypes. In other embodiments,the immune response induced by an active compound or a compositiondescribed herein is not effective to prevent and/or treat an influenzavirus infection caused by H3N2 subtypes.

In some embodiments, the immune response induced by an active compoundor a composition described herein is effective to prevent and/or treatan influenza virus disease caused by any subtype or strain of influenzavirus. In certain embodiments, the immune response induced by an activecompound or a composition described herein is effective to preventand/or treat an influenza virus disease caused by a subtype of influenzavirus that belongs to one HA group and not the other HA group. Forexample, the immune response induced may be effective to prevent and/ortreat an influenza virus disease caused by an influenza virus thatbelongs to the HA group consisting of H11, H13, H16, H9, H8, H12, H6,H1, H5 and H2. Alternatively, the immune response induced may beeffective to prevent and/or treat an influenza virus disease caused byan influenza virus that belongs to the HA group consisting of H3, H4,H14, H10, H15 and H7. In some embodiments, the immune response inducedby an active compound or a composition described herein is effective toprevent and/or treat an influenza virus disease caused by any of one,two, three, four or five subtypes of influenza virus. In certainembodiments, the immune response induced by an active compound or acomposition described herein is effective to prevent and/or treat aninfluenza virus disease caused by any of six, seven, eight, nine, ten,eleven, twelve, thirteen, fourteen or fifteen subtypes of influenzavirus. In some embodiments, the immune response induced by an activecompound or a composition described herein is effective to preventand/or treat an influenza virus disease caused by one or more variantswithin the same subtype of influenza virus.

In some embodiments, the immune response induced by an active compoundor a composition described herein is effective to reduce symptomsresulting from an influenza virus disease/infection. Symptoms ofinfluenza virus disease/infection include, but are not limited to, bodyaches (especially joints and throat), fever, nausea, headaches,irritated eyes, fatigue, sore throat, reddened eyes or skin, andabdominal pain.

In some embodiments, the immune response induced by an active compoundor a composition described herein is effective to reduce thehospitalization of a subject suffering from an influenza virusdisease/infection. In some embodiments, the immune response induced byan active compound or a composition described herein is effective toreduce the duration of hospitalization of a subject suffering from aninfluenza virus disease/infection.

In another aspect, provided herein are methods for preventing and/ortreating an influenza virus infection in a subject utilizing an activecompound (e.g., an influenza hemagglutinin stem domain polypeptidedescribed herein, a nucleic acid encoding such a polypeptide, a vectorcontaining or expressing such a polypeptide, or cells stimulated withsuch a polypeptide) or a composition described herein. In oneembodiment, a method for preventing or treating an influenza virusinfection in a subject comprises administering to a subject in needthereof an influenza hemagglutinin stem domain polypeptide, a nucleicacid encoding such a polypeptide, a vector containing or expressing sucha polypeptide, or a composition of any one of the foregoing. In aspecific embodiment, a method for preventing or treating an influenzavirus infection in a subject comprises administering to a subject inneed thereof a subunit vaccine, a live virus vaccine, an inactivatedvirus vaccine, a split virus vaccine or a viral-like particle vaccine.

In another aspect, provided herein are methods for preventing and/ortreating an influenza virus disease in a subject utilizing an influenzahemagglutinin stem domain polypeptide described herein, a nucleic acidencoding such a polypeptide, a vector containing or expressing such apolypeptide, or cells stimulated with such a polypeptide. In a specificembodiment, a method for preventing or treating an influenza virusdisease in a subject comprises administering to a subject in needthereof an effective amount of an influenza hemagglutinin stem domainpolypeptide or an immunogenic composition thereof. In anotherembodiment, a method for preventing or treating an influenza virusdisease in a subject comprises administering to a subject in needthereof an effective amount of a nucleic acid encoding an influenzahemagglutinin stem domain polypeptide or an immunogenic compositionthereof. In another embodiment, a method for preventing or treating aninfluenza virus disease in a subject comprises administering to asubject in need thereof an effective amount of a viral vector containingor expressing an influenza hemagglutinin stem domain polypeptide or animmunogenic composition thereof. In yet another embodiment, a method forpreventing or treating an influenza virus disease in a subject comprisesadministering to a subject in need thereof an effective amount of cellsstimulated with an influenza hemagglutinin stem domain polypeptide or apharmaceutical composition thereof.

In a specific embodiment, a method for preventing or treating aninfluenza virus disease in a subject comprises administering to asubject in need thereof a subunit vaccine described herein. In anotherembodiment, a method for preventing or treating an influenza virusdisease in a subject comprises administering to a subject in needthereof a live virus vaccine described herein. In particularembodiments, the live virus vaccine comprises an attenuated virus. Inanother embodiment, a method for preventing or treating an influenzavirus disease in a subject comprises administering to a subject in needthereof an inactivated virus vaccine described herein. In anotherembodiment, a method for preventing or treating an influenza virusdisease in a subject comprises administering to a subject in needthereof a split virus vaccine described herein. In another embodiment, amethod for preventing or treating an influenza virus disease comprisesadministering to a subject in need thereof a viral-like particle vaccinedescribed herein. In another embodiment, a method for preventing ortreating an influenza virus disease in a subject, comprisingadministering to a subject in need thereof a virosome described herein.In another embodiment, a method for preventing or treating an influenzavirus disease in a subject comprising administering to a subject in needthereof a bacteria expressing or engineered to express an influenzahemagglutinin stem domain polypeptide or a composition thereof.

In another aspect, provided herein are methods of preventing and/ortreating an influenza virus disease in a subject by administeringneutralizing antibodies described herein. In a specific embodiment, amethod for preventing or treating an influenza virus disease in asubject comprises administering to a subject in need thereof aneffective amount of a neutralizing antibody described herein, or apharmaceutical composition thereof. In particular embodiments, theneutralizing antibody is a monoclonal antibody. In certain embodiments,the neutralizing antibody is not CR6261, CR6325, CR6329, CR6307, CR6323,2A, D7, D8, F10, G17, H40, A66, D80, E88, E90, H98, C179 (FERM BP-4517),AI3C (FERM BP-4516) or any other antibody described in Ekiert D C et al.(2009) Antibody Recognition of a Highly Conserved Influenza VirusEpitope. Science (published in Science Express Feb. 26, 2009); Kashyapet al. (2008) Combinatorial antibody libraries from survivors of theTurkish H5N1 avian influenza outbreak reveal virus neutralizationstrategies. Proc Natl Acad Sci USA 105: 5986-5991; Sui et al. (2009)Structural and functional bases for broad-spectrum neutralization ofavian and human influenza A viruses. Nat Struct Mol Biol 16: 265-273;U.S. Pat. Nos. 5,589,174, 5,631,350, 6,337,070, and 6,720,409;International Application No. PCT/US2007/068983 published asInternational Publication No. WO 2007/134237; International ApplicationNo. PCT/US2008/075998 published as International Publication No. WO2009/036157; International Application No. PCT/EP2007/059356 publishedas International Publication No. WO 2008/028946; and InternationalApplication No. PCT/US2008/085876 published as International PublicationNo. WO 2009/079259. In other embodiments, the neutralizing antibody isnot an antibody described in Wang et al. (2010) “Broadly ProtectiveMonoclonal Antibodies against H3 Influenza Viruses following SequentialImmunization with Different Hemagglutinins,” PLOS Pathogens 6(2):1-9.

In certain embodiments, the methods for preventing or treating aninfluenza virus disease or infection in a subject (e.g., a human ornon-human animal) provided herein result in a reduction in thereplication of the influenza virus in the subject as measured by in vivoand in vitro assays known to those of skill in the art and describedherein. In some embodiments, the replication of the influenza virus isreduced by approximately 1 log or more, approximately 2 logs or more,approximately 3 logs or more, approximately 4 logs or more,approximately 5 logs or more, approximately 6 logs or more,approximately 7 logs or more, approximately 8 logs or more,approximately 9 logs or more, approximately 10 logs or more, 1 to 3logs, 1 to 5 logs, 1 to 8 logs, 1 to 9 logs, 2 to 10 logs, 2 to 5 logs,2 to 7 logs, 2 logs to 8 logs, 2 to 9 logs, 2 to 10 logs 3 to 5 logs, 3to 7 logs, 3 to 8 logs, 3 to 9 logs, 4 to 6 logs, 4 to 8 logs, 4 to 9logs, 5 to 6 logs, 5 to 7 logs, 5 to 8 logs, 5 to 9 logs, 6 to 7 logs, 6to 8 logs, 6 to 9 logs, 7 to 8 logs, 7 to 9 logs, or 8 to 9 logs.

5.12.1 Combination Therapies

In various embodiments, an influenza hemagglutinin stem domainpolypeptide described herein, a nucleic acid encoding such apolypeptide, a vector (e.g., a viral vector or a bacteria) containing orexpressing such a polypeptide, cells stimulated with such a polypeptide,or a neutralizing antibody may be administered to a subject incombination with one or more other therapies (e.g., antiviral,antibacterial, or immunomodulatory therapies). In some embodiments, apharmaceutical composition (e.g., an immunogenic composition) describedherein may be administered to a subject in combination with one or moretherapies. The one or more other therapies may be beneficial in thetreatment or prevention of an influenza virus disease or may amelioratea symptom or condition associated with an influenza virus disease. Insome embodiments, the one or more other therapies are pain relievers,anti-fever medications, or therapies that alleviate or assist withbreathing. In certain embodiments, the therapies are administered lessthan 5 minutes apart, less than 30 minutes apart, 1 hour apart, at about1 hour apart, at about 1 to about 2 hours apart, at about 2 hours toabout 3 hours apart, at about 3 hours to about 4 hours apart, at about 4hours to about 5 hours apart, at about 5 hours to about 6 hours apart,at about 6 hours to about 7 hours apart, at about 7 hours to about 8hours apart, at about 8 hours to about 9 hours apart, at about 9 hoursto about 10 hours apart, at about 10 hours to about 11 hours apart, atabout 11 hours to about 12 hours apart, at about 12 hours to 18 hoursapart, 18 hours to 24 hours apart, 24 hours to 36 hours apart, 36 hoursto 48 hours apart, 48 hours to 52 hours apart, 52 hours to 60 hoursapart, 60 hours to 72 hours apart, 72 hours to 84 hours apart, 84 hoursto 96 hours apart, or 96 hours to 120 hours part. In specificembodiments, two or more therapies are administered within the samepatent visit.

Any anti-viral agents well-known to one of skill in the art may used incombination with an active compound or pharmaceutical compositiondescribed herein. Non-limiting examples of anti-viral agents includeproteins, polypeptides, peptides, fusion proteins antibodies, nucleicacid molecules, organic molecules, inorganic molecules, and smallmolecules that inhibit and/or reduce the attachment of a virus to itsreceptor, the internalization of a virus into a cell, the replication ofa virus, or release of virus from a cell. In particular, anti-viralagents include, but are not limited to, nucleoside analogs (e.g.,zidovudine, acyclovir, gangcyclovir, vidarabine, idoxuridine,trifluridine, and ribavirin), foscarnet, amantadine, peramivir,rimantadine, saquinavir, indinavir, ritonavir, alpha-interferons andother interferons, AZT, zanamivir (Relenza®), and oseltamivir(Tamiflu®). Other anti-viral agents include influenza virus vaccines,e.g., Fluarix® (GlaxoSmithKline), FluMist® (MedImmune Vaccines),Fluvirin® (Chiron Corporation), Flulaval® (GlaxoSmithKline), Afluria®(CSL Biotherapies Inc.), Agriflu® (Novartis) or Fluzone® (AventisPasteur).

In specific embodiments, the anti-viral agent is an immunomodulatoryagent that is specific for a viral antigen. In particular embodiments,the viral antigen is an influenza virus polypeptide other than ahemagglutinin polypeptide. In other embodiments, the viral antigen is aninfluenza virus hemagglutinin polypeptide.

Any anti-bacterial agents known to one of skill in the art may used incombination with an active compound or pharmaceutical compositiondescribed herein. Non-limiting examples of anti-bacterial agents includeAmikacin, Amoxicillin,

Amoxicillin-clavulanic acid, Amphothericin-B, Ampicillin,Ampicllin-sulbactam, Apramycin, Azithromycin, Aztreonam, Bacitracin,Benzylpenicillin, Caspofungin, Cefaclor, Cefadroxil, Cefalexin,Cefalothin, Cefazolin, Cefdinir, Cefepime, Cefixime, Cefmenoxime,Cefoperazone, Cefoperazone-sulbactam, Cefotaxime, Cefoxitin, Cefpirome,Cefpodoxime, Cefpodoxime-clavulanic acid, Cefpodoxime-sulbactam,Cefprozil, Cefquinome, Ceftazidime, Ceftibutin, Ceftiofur, Ceftobiprole,Ceftriaxon, Cefuroxime, Chloramphenicole, Florfenicole, Ciprofloxacin,Clarithromycin, Clinafloxacin, Clindamycin, Cloxacillin, Colistin,Cotrimoxazol (Trimthoprim/sulphamethoxazole), Dalbavancin,Dalfopristin/Quinopristin, Daptomycin, Dibekacin, Dicloxacillin,Doripenem, Doxycycline, Enrofloxacin, Ertapenem, Erythromycin,Flucloxacillin, Fluconazol, Flucytosin, Fosfomycin, Fusidic acid,Garenoxacin, Gatifloxacin, Gemifloxacin, Gentamicin, Imipenem,Itraconazole, Kanamycin, Ketoconazole, Levofloxacin, Lincomycin,Linezolid, Loracarbef, Mecillnam (amdinocillin), Meropenem,Metronidazole, Meziocillin, Mezlocillin-sulbactam, Minocycline,Moxifloxacin, Mupirocin, Nalidixic acid, Neomycin, Netilmicin,Nitrofurantoin, Norfloxacin, Ofloxacin, Oxacillin, Pefloxacin,Penicillin V, Piperacillin, Piperacillin-sulbactam,Piperacillin-tazobactam, Rifampicin, Roxythromycin, Sparfloxacin,Spectinomycin, Spiramycin, Streptomycin, Sulbactam, Sulfamethoxazole,Teicoplanin, Telavancin, Telithromycin, Temocillin, Tetracyklin,Ticarcillin, Ticarcillin-clavulanic acid, Tigecycline, Tobramycin,Trimethoprim, Trovafloxacin, Tylosin, Vancomycin, Virginiamycin, andVoriconazole.

In some embodiments, a combination therapy comprises active immunizationwith an influenza hemagglutinin stem domain polypeptide, or one or morevectors described in Sections 5.2-5.7 and passive immunization with oneor more neutralizing antibodies described in Section 5.9. In someembodiments, a combination therapy comprises immunization with one ormore vectors described in Sections 5.2-5.7 and administration of cells(e.g., by adoptive transfer) described in Section 5.9.

In some embodiments, a combination therapy comprises administration oftwo or more different vectors described in Sections 5.2-5.7. In oneexample, one or more vectors expressing an influenza hemagglutinin stemdomain polypeptide derived from an influenza A virus hemagglutininpolypeptide and one or more vectors expressing an influenzahemagglutinin stem domain polypeptide derived from an influenza B virushemagglutinin polypeptide are administered in combination. In someembodiments, a combination therapy comprises administration of a vectorexpressing an influenza hemagglutinin stem domain polypeptide derivedfrom an influenza A virus H3 antigen and a vector expressing aninfluenza hemagglutinin stem domain polypeptide derived from aninfluenza A virus H1 antigen. In some embodiments, the combinationtherapy comprises administration of a vector expressing an influenzahemagglutinin stem domain polypeptide derived from an influenza A virusH3 antigen, a vector expressing an influenza hemagglutinin stem domainpolypeptide derived from an influenza A virus H1 antigen, and a vectorexpressing an influenza hemagglutinin stem domain polypeptide derivedfrom an influenza B virus hemagglutinin polypeptide.

In some embodiments, a combination therapy comprises active immunizationwith an active compound that induces an immune response to one, two,three, or more HA subtypes in one HA group (e.g., Group 1) incombination with an active compound that induces an immune response toone, two, three, or more HA subtypes in the other HA group (e.g., Group2).

In some embodiments, a combination therapy comprises active immunizationwith two or more influenza hemagglutinin stem domain polypeptidesdescribed in Section 5.1.

In certain embodiments, a combination therapy comprises activeimmunization with one, two, or more influenza hemagglutinin stem domainpolypeptides derived from an influenza A virus and one or more influenzahemagglutinin stem domain polypeptides derived from an influenza Bvirus.

5.12.2 Patient Populations

In certain embodiments, an active compound or composition describedherein may be administered to a naïve subject, i.e., a subject that doesnot have a disease caused by influenza virus infection or has not beenand is not currently infected with an influenza virus infection. In oneembodiment, an active compound or composition described herein isadministered to a naïve subject that is at risk of acquiring aninfluenza virus infection. In one embodiment, an active compound orcomposition described herein is administered to a subject that does nothave a disease caused by the specific influenza virus, or has not beenand is not infected with the specific influenza virus to which theinfluenza hemagglutinin stem domain polypeptide induces an immuneresponse. An active compound or composition described herein may also beadministered to a subject that is and/or has been infected with theinfluenza virus or another type, subtype or strain of the influenzavirus to which the influenza hemagglutinin stem domain polypeptideinduces an immune response.

In certain embodiments, an active compound or composition describedherein is administered to a patient who has been diagnosed with aninfluenza virus infection. In some embodiments, an active compound orcomposition described herein is administered to a patient infected withan influenza virus before symptoms manifest or symptoms become severe(e.g., before the patient requires hospitalization). In someembodiments, an active compound or composition described herein isadministered to a patient that is infected with or has been diagnosedwith a different type of influenza virus than that of the influenzavirus from which the HA stem domain polypeptide of the active compoundor composition was derived.

In certain embodiments, an active compound or composition describedherein is administered to a patient that may be or is infected with aninfluenza virus that belongs to the same HA group as that of theinfluenza hemagglutinin stem domain polypeptide. In certain embodiments,an active compound or composition described herein is administered to apatient that may be or is infected with an influenza virus of the samesubtype as that of the influenza hemagglutinin stem domain polypeptide.

In some embodiments, a subject to be administered an active compound orcomposition described herein is an animal. In certain embodiments, theanimal is a bird. In certain embodiments, the animal is a canine. Incertain embodiments, the animal is a feline. In certain embodiments, theanimal is a horse. In certain embodiments, the animal is a cow. Incertain embodiments, the animal is a mammal, e.g., a horse, swine,mouse, or primate, preferably a human.

In certain embodiments, a subject to be administered an active compoundor composition described herein is a human adult. In certainembodiments, a subject to be administered an active compound orcomposition described herein is a human adult more than 50 years old. Incertain embodiments, a subject to be administered an active compound orcomposition described herein is an elderly human subject.

In certain embodiments, a subject to be administered an active compoundor composition described herein is a human child. In certainembodiments, a subject to be administered an active compound orcomposition described herein is a human infant. In certain embodiments,a subject to whom an active compound or composition described herein isadministered is not an infant of less than 6 months old. In a specificembodiment, a subject to be administered an active compound orcomposition described herein is 2 years old or younger.

In specific embodiments, a subject to be administered an active compoundor composition described herein is any infant or child more than 6months of age and any adult over 50 years of age. In other embodiments,the subject is an individual who is pregnant. In another embodiment, thesubject is an individual who may or will be pregnant during theinfluenza season (e.g., November to April). In specific embodiments, asubject to be administered an active compound or composition describedherein is a woman who has given birth 1, 2, 3, 4, 5, 6, 7, or 8 weeksearlier.

In some embodiments, the human subject to be administered an activecompound or composition described herein is any individual at increasedrisk of influenza virus infection or disease resulting from influenzavirus infection (e.g., an immunocompromised or immunodeficientindividual). In some embodiments, the human subject to be administeredan active compound or composition described herein is any individual inclose contact with an individual with increased risk of influenza virusinfection or disease resulting from influenza virus infection (e.g.,immunocompromised or immunosuppressed individuals).

In some embodiments, the human subject to be administered an activecompound or composition described herein is an individual affected byany condition that increases susceptibility to influenza virus infectionor complications or disease resulting from influenza virus infection. Inother embodiments, an active compound or composition described herein isadministered to a subject in which an influenza virus infection has thepotential to increase complications of another condition that theindividual is affected by, or for which they are at risk. In particularembodiments, such conditions that increase susceptibility to influenzavirus complications or for which influenza virus increases complicationsassociated with the condition are, e.g., conditions that affect thelung, such as cystic fibrosis, emphysema, asthma, or bacterialinfections (e.g., infections caused by Haemophilus influenzae,Streptococcus pneumoniae, Legionella pneumophila, and Chlamydiatrachomatus); cardiovascular disease (e.g., congenital heart disease,congestive heart failure, and coronary artery disease); endocrinedisorders (e.g., diabetes), neurological and neuron-developmentalconditions (e.g., disorders of the brain, the spinal cord, theperipheral nerve, and muscle (such as cerebral palsy, epilepsy (seizuredisorders), stroke, intellectual disability (e.g., mental retardation),muscular dystrophy, and spinal cord injury)).

In some embodiments, the human subject to be administered an activecompound or composition described herein is an individual that residesin a group home, such as a nursing home. In some embodiments, the humansubject to be administered an active compound or composition describedherein works in, or spends a significant amount of time in, a grouphome, e.g., a nursing home. In some embodiments, the human subject to beadministered an active compound or composition described herein is ahealth care worker (e.g., a doctor or nurse). In some embodiments, thehuman subject to be administered an active compound or compositiondescribed herein is a smoker. In a specific embodiment, the humansubject to be administered an active compound or composition describedherein is immunocompromised or immunosuppressed.

In addition, subjects at increased risk of developing complications frominfluenza who may be administered an active compound or compositiondescribed herein include: any individual who can transmit influenzaviruses to those at high risk for complications, such as, e.g., membersof households with high-risk individuals, including households that willinclude infants younger than 6 months, individuals coming into contactwith infants less than 6 months of age, or individuals who will comeinto contact with individuals who live in nursing homes or otherlong-term care facilities; individuals with long-term disorders of thelungs, heart, or circulation; individuals with metabolic diseases (e.g.,diabetes); individuals with kidney disorders; individuals with blooddisorders (including anemia or sickle cell disease); individuals withweakened immune systems (including immunosuppression caused bymedications, malignancies such as cancer, organ transplant, or HIVinfection); children who receive long-term aspirin therapy (andtherefore have a higher chance of developing Reye syndrome if infectedwith influenza).

In other embodiments, subjects for administration of an active compoundor composition described herein include healthy individuals six monthsof age or older, who: plan to travel to foreign countries and areaswhere flu outbreaks may be occurring, such, e.g., as the tropics and theSouthern Hemisphere from April through September; travel as a part oflarge organized tourist groups that may include persons from areas ofthe world where influenza viruses are circulating; attend school orcollege and reside in dormitories, or reside in institutional settings;or wish to reduce their risk of becoming ill with influenza.

In some embodiments, a subject for whom administration of an activecompound or composition described herein is contraindicated include anyindividual for whom influenza vaccination is contraindicated, such as:infants younger than six months of age; and individuals who have had ananaphylactic reaction (allergic reactions that cause difficultybreathing, which is often followed by shock) to eggs, egg products, orother components used in the production of the immunogenic formulation.In certain embodiments, when administration of an active compound orcomposition described herein is contraindicated due to one or morecomponents used in the production of the immunogenic formulation (e.g.,due to the presence of egg or egg products), the active compound orcomposition may be produced in a manner that does not include thecomponent that causes the administration of an active compound orcomposition to be contraindicated (e.g., the active compound orcomposition may be produced without the use of eggs or egg products).

In some embodiments, it may be advisable not to administer a live virusvaccine to one or more of the following patient populations: elderlyhumans; infants younger than 6 months old; pregnant individuals; infantsunder the age of 1 years old; children under the age of 2 years old;children under the age of 3 years old; children under the age of 4 yearsold; children under the age of 5 years old; adults under the age of 20years old; adults under the age of 25 years old; adults under the age of30 years old; adults under the age of 35 years old; adults under the ageof 40 years old; adults under the age of 45 years old; adults under theage of 50 years old; elderly humans over the age of 70 years old;elderly humans over the age of 75 years old; elderly humans over the ageof 80 years old; elderly humans over the age of 85 years old; elderlyhumans over the age of 90 years old; elderly humans over the age of 95years old; children and adolescents (2-17 years of age) receivingaspirin or aspirin-containing medications, because of the complicationsassociated with aspirin and wild-type influenza virus infections in thisage group; individuals with a history of asthma or other reactive airwaydiseases; individuals with chronic underlying medical conditions thatmay predispose them to severe influenza infections; individuals with ahistory of Guillain-Barre syndrome; individuals with acute seriousillness with fever; or individuals who are moderately or severely ill.For such individuals, administration of inactivated virus vaccines,split virus vaccines, subunit vaccines, virosomes, viral-like particlesor the non-viral vectors described herein may be preferred. In certainembodiments, subjects preferably administered a live virus vaccine mayinclude healthy children and adolescents, ages 2-17 years, and healthyadults, ages 18-49.

In certain embodiments, an immunogenic formulation comprising a livevirus vector is not given concurrently with other live-virus vaccines.

5.13 Modes of Administration

5.13.1 Routes of Delivery

An active compound or composition described herein may be delivered to asubject by a variety of routes. These include, but are not limited to,intranasal, intratracheal, oral, intradermal, intramuscular,intraperitoneal, transdermal, intravenous, conjunctival and subcutaneousroutes. In some embodiments, a composition is formulated for topicaladministration, for example, for application to the skin. In specificembodiments, the route of administration is nasal, e.g., as part of anasal spray. In certain embodiments, a composition is formulated forintramuscular administration. In some embodiments, a composition isformulated for subcutaneous administration. In certain embodiments, acomposition is not formulated for administration by injection. Inspecific embodiments for live virus vaccines, the vaccine is formulatedfor administration by a route other than injection.

In cases where the antigen is a viral vector, a virus-like particlevector, or a bacterial vector, for example, it may be preferable tointroduce an immunogenic composition via the natural route of infectionof the backbone virus or bacteria from which the vector was derived.Alternatively, it may be preferable to introduce an influenzahemagglutinin stem domain polypeptide via the natural route of infectionof the influenza virus from which polypeptide is derived. The ability ofan antigen, particularly a viral vector, to induce a vigorous secretoryand cellular immune response can be used advantageously. For example,infection of the respiratory tract by a viral vector may induce a strongsecretory immune response, for example in the urogenital system, withconcomitant protection against an influenza virus. In addition, in apreferred embodiment it may be desirable to introduce the pharmaceuticalcompositions into the lungs by any suitable route. Pulmonaryadministration can also be employed, e.g., by use of an inhaler ornebulizer, and formulation with an aerosolizing agent for use as aspray.

In a specific embodiment, a subunit vaccine is administeredintramuscularly. In another embodiment, a live influenza virus or liveNDV vaccine is administered intranasally. In another embodiment, aninactivated influenza virus vaccine, or a split influenza virus vaccineis administered intramuscularly. In another embodiment, an inactivatedNDV virus vaccine or a split NDV virus vaccine is administeredintramuscularly. In another embodiment, a viral-like particle orcomposition thereof is administered intramuscularly.

In some embodiments, cells stimulated with an influenza hemagglutininstem domain polypeptide in vitro may be introduced (or re-introduced)into a subject using techniques known to one of skill in the art. Insome embodiments, the cells can be introduced into the dermis, under thedermis, or into the peripheral blood stream. In some embodiments, thecells introduced into a subject are preferably cells derived from thatsubject, to avoid an adverse immune response. In other embodiments,cells also can be used that are derived from a donor host having asimilar immune background. Other cells also can be used, including thosedesigned to avoid an adverse immunogenic response.

5.13.2 Dosage and Frequency of Administration

The amount of an active compound or composition which will be effectivein the treatment and/or prevention of an influenza virus infection or aninfluenza virus disease will depend on the nature of the disease, andcan be determined by standard clinical techniques.

The precise dose to be employed in the formulation will also depend onthe route of administration, and the seriousness of the infection ordisease caused by it, and should be decided according to the judgment ofthe practitioner and each subject's circumstances. For example,effective doses may also vary depending upon means of administration,target site, physiological state of the patient (including age, bodyweight, health), whether the patient is human or an animal, othermedications administered, and whether treatment is prophylactic ortherapeutic. Usually, the patient is a human but nonhuman mammalsincluding transgenic mammals can also be treated. Treatment dosages areoptimally titrated to optimize safety and efficacy.

In certain embodiments, an in vitro assay is employed to help identifyoptimal dosage ranges. Effective doses may be extrapolated from doseresponse curves derived from in vitro or animal model test systems.

Exemplary doses for nucleic acids encoding influenza hemagglutinin stemdomain polypeptides range from about 10 ng to 1 g, 100 ng to 100 mg, 1μg to 10 mg, or 30-300 μg nucleic acid, e.g., DNA, per patient.

In certain embodiments, exemplary doses for influenza hemagglutinin stemdomain polypeptides (e.g., as provided in split virus vaccines andsubunit vaccines) range from about 5 μg to 100 mg, 15 μg to 50 mg, 15 μgto 25 mg, 15 μg to 10 mg, 15 μg to 5 mg, 15 μg to 1 mg, 15 μg to 100 μg,15 μg to 75 μg, 5 μg to 50 μg, 10 μg to 50 μg, 15 μg to 45 μg, 20 μg to40 μg, or 25 to 35 μg per kilogram of the patient. In other embodiments,exemplary doses for influenza hemagglutinin stem domain polypeptidesrange from about 1 μg to 50 mg, 5 μg to 50 mg, 1 μg to 100 mg, 5 μg to100 mg, 15 μg to 50 mg, 15 μg to 25 mg, 15 μg to 10 mg, 15 μg to 5 mg,15 μg to 1 mg, 15 μg to 100 μg, 15 μg to 75 μg, 5 μg to 50 μg, 10 μg to50 μg, 15 μg to 45 μg, 20 μg to 40 μg, or 25 to 35 μg of influenzahemagglutinin stem domain polypeptides per dose.

Doses for infectious viral vectors may vary from 10-100, or more,virions per dose. In some embodiments, suitable dosages of a virusvector are 10², 5×10², 10³, 5×10³, 10⁴ 5×10⁴ 10⁵ 5×10⁵ 10⁶ 5×10⁶ 10⁷5×10⁷ 10⁸ 5×10⁸ 1×10⁹ 5×10⁹ 1×10¹⁰, 5×10¹⁰, 1×10¹¹, 5×10¹¹ or 10¹² pfu,and can be administered to a subject once, twice, three or more timeswith intervals as often as needed.

In certain embodiments, exemplary doses for VLPs range from about 0.01μg to about 100 mg, about 0.1 μg to about 100 mg, about 5 μg to about100 mg, about 15 μg to about 50 mg, about 15 μg to about 25 mg, about 15μg to about 10 mg, about 15 μg to about 5 mg, about 15 μg to about 1 mg,about 15 μg to about 100 μg, about 15 μg to about 75 μg, about 5 μg toabout 50 μg, about 10 μg to about 50 μg, about 15 μg to about 45 μg,about 20 μg to about 40 or about 25 to about 35 μg per kilogram of thepatient. In other embodiments, exemplary doses for influenzahemagglutinin stem domain polypeptides range from about 1 μg to about 50mg, about 5 μg to about 50 mg, about 1 μg to about 100 mg, about 5 μg toabout 100 mg, about 15 μg to about 50 mg, about 15 μg to about 25 mg,about 15 μg to about 10 mg, about 15 μg to about 5 mg, about 15 μg toabout 1 mg, about 15 μg to about 100 μg, about 15 μg to about 75 μg,about 5 μg to about 50 μg, about 10 μg to about 50 μg, about 15 μg toabout 45 μg, about 20 μg to about 40 μg, or about 25 to about 35 μg ofinfluenza hemagglutinin stem domain polypeptides per dose, and can beadministered to a subject once, twice, three or more times withintervals as often as needed.

In one embodiment, an inactivated vaccine is formulated such that itcontains about 5 μg to about 50 μg, about 10 μg to about 50 μg, aboutabout 15 μg to about 100 μg, about 15 μg to about 75 μg, about 15 μg toabout 50 μg, about 15 μg to about 30 μg, about 20 μg to about 50 μg,about 25 μg to about 40 μg, about 25 μg to about 35 μg of an influenzahemagglutinin stem domain polypeptide. Such a vaccine may contain acombination of one or more different influenza hemagglutinin stem domainpolypeptides, for example, one or more influenza hemagglutinin stemdomain polypeptides from an influenza A virus and one or more influenzahemagglutinin stem domain polypeptides from an influenza B virus. Insome embodiments, influenza hemagglutinin stem domain polypeptidesderived from, e.g., A/H1N1, A/H3N2, and B hemagglutinin polypeptides areincluded in a trivalent inactivated vaccine (TIV), formulated such thata 0.5-mL dose contains 15 μg each of influenza hemagglutinin stem domainpolypeptide. In one embodiment, a live attenuated influenza vaccine(LAIV) is formulated such that a 0.2-mL dose contains 10^(6.5-7.5)fluorescent focal units of live attenuated influenza viruses from threestrains expressing at least one influenza hemagglutinin stem domainpolypeptide.

In certain embodiments, an active compound or composition isadministered to a subject once as a single dose. In certain embodiments,an active compound or composition is administered to a subject as asingle dose followed by a second dose 3 to 6 weeks later. In accordancewith these embodiments, booster inoculations may be administered to thesubject at 6 to 12 month intervals following the second inoculation. Incertain embodiments, the booster inoculations may utilize a differentactive compound or composition. In some embodiments, the administrationof the same active compound or composition may be repeated and theadministrations may be separated by at least 1 day, 2 days, 3 days, 5days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months,or at least 6 months. In certain embodiments, an active compound orcomposition is administered to a subject as a single dose once per year.

In specific embodiments for administration to children, two doses of anactive compound or composition, given at least one month apart, areadministered to a child. In specific embodiments for administration toadults, a single dose is given. In another embodiment, two doses of anactive compound or composition, given at least one month apart, areadministered to an adult. In another embodiment, a young child (sixmonths to nine years old) may be administered an active compound orcomposition for the first time in two doses given one month apart. In aparticular embodiment, a child who received only one dose in their firstyear of vaccination should receive two doses in the following year. Insome embodiments, two doses administered 4 weeks apart are preferred forchildren 2-8 years of age who are administered an influenza vaccine,e.g., an immunogenic formulation described herein, for the first time.In certain embodiments, for children 6-35 months of age, a half dose(0.25 ml) may be preferred, in contrast to 0.5 ml which may be preferredfor subjects over three years of age.

In particular embodiments, an active compound or composition isadministered to a subject in the fall or winter, i.e., prior to orduring the influenza season in each hemisphere. In one embodiment,children are administered their first dose early in the season, e.g.,late September or early October, so that the second dose can be givenprior to the peak of the influenza season.

For passive immunization with an antibody, the dosage ranges from about0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the patientbody weight. For example, dosages can be 1 mg/kg body weight or 10 mg/kgbody weight or within the range of 1-10 mg/kg or in other words, 70 mgor 700 mg or within the range of 70-700 mg, respectively, for a 70 kgpatient. An exemplary treatment regime entails administration once perevery two weeks or once a month or once every 3 to 6 months for a periodof one year or over several years, or over several year-intervals. Insome methods, two or more monoclonal antibodies with different bindingspecificities are administered simultaneously, in which case the dosageof each antibody administered falls within the ranges indicated.

Antibody is usually administered on multiple occasions. Intervalsbetween single dosages can be weekly, monthly or yearly. Intervals canalso be irregular as indicated by measuring blood levels of antibody tothe influenza hemagglutinin stem domain polypeptide in the patient.

5.14 Biological Assays

5.14.1 Assays for Testing Activity of Influenza Hemagglutinin StemDomain Polypeptide

Assays for testing the expression of a influenza hemagglutinin stemdomain polypeptide in a vector disclosed herein may be conducted usingany assay known in the art. For example, an assay for incorporation intoa viral vector comprises growing the virus as described in this sectionor Sections 5.4 or 5.5, purifying the viral particles by centrifugationthrough a sucrose cushion, and subsequent analysis for influenzahemagglutinin stem domain polypeptide expression by an immunoassay, suchas Western blotting, using methods well known in the art.

In one embodiment, an influenza hemagglutinin stem domain polypeptidedisclosed herein is assayed for proper folding and functionality bytesting its ability to bind specifically to a neutralizing antibodydirected to an influenza virus hemagglutinin polypeptide, such as thestalk region of the polypeptide, using any assay for antibody-antigeninteraction known in the art. Neutralizing antibodies for use in suchassays include, for example, the neutralizing antibodies described inEkiert et al., 2009, Science Express, 26 Feb. 2009; Kashyap et al.,2008, Proc Natl Acad Sci USA 105: 5986-5991; Sui et al. 2009, NatureStructural and Molecular Biology, 16:265-273; Wang et al., 2010, PLOSPathogens 6(2):1-9; U.S. Pat. Nos. 5,589,174, 5,631,350, 6,337,070, and6,720,409; International Application No. PCT/US2007/068983 published asInternational Publication No. WO 2007/134237; International ApplicationNo. PCT/US2008/075998 published as International Publication No. WO2009/036157; International Application No. PCT/EP2007/059356 publishedas International Publication No. WO 2008/028946; and InternationalApplication No. PCT/US2008/085876 published as International PublicationNo. WO 2009/079259. These antibodies include CR6261, CR6325, CR6329,CR6307, CR6323, 2A, D7, D8, F10, G17, H40, A66, D80, E88, E90, H98, C179(FERM BP-4517), AI3C (FERM BP-4516), among others.

In another embodiment, an influenza hemagglutinin stem domainpolypeptide disclosed herein is assayed for proper folding bydetermination of the structure or conformation of the influenzahemagglutinin stem domain polypeptide using any method known in the artsuch as, e.g., NMR, X-ray crystallographic methods, or secondarystructure prediction methods, e.g., circular dichroism.

5.14.2 Assays for Testing Activity of Antibodies Generated UsingInfluenza Hemagglutinin Stem Domain Polypeptide

Antibodies described herein may be characterized in a variety of waysknown to one of skill in the art (e.g. ELISA, Surface Plasmon resonancedisplay (BIAcore), Western blot, immunofluorescence, immunostainingand/or microneutralization assays). In some embodiments, antibodies areassayed for the ability to specifically bind to an influenza virushemagglutinin polypeptide, or a vector comprising said polypeptide. Suchan assay may be performed in solution (e.g., Houghten, 1992,Bio/Techniques 13:412 421), on beads (Lam, 1991, Nature 354:82 84), onchips (Fodor, 1993, Nature 364:555 556), on bacteria (U.S. Pat. No.5,223,409), on spores (U.S. Pat. Nos. 5,571,698; 5,403,484; and5,223,409), on plasmids (Cull et al., 1992, Proc. Natl. Acad. Sci. USA89:1865 1869) or on phage (Scott and Smith, 1990, Science 249:386 390;Cwirla et al., 1990, Proc. Natl. Acad. Sci. USA 87:6378 6382; andFelici, 1991, J. Mol. Biol. 222:301 310) (each of these references isincorporated herein in its entirety by reference).

Specific binding of an antibody to the influenza virus hemagglutininpolypeptide and cross-reactivity with other antigens can be assessed byany method known in the art. Immunoassays which can be used to analyzespecific binding and cross-reactivity include, but are not limited to,competitive and non-competitive assay systems using techniques such aswestern blots, radioimmunoassays, ELISA (enzyme linked immunosorbentassay), “sandwich” immunoassays, immunoprecipitation assays, precipitinreactions, gel diffusion precipitin reactions, immunodiffusion assays,agglutination assays, complement-fixation assays, immunoradiometricassays, fluorescent immunoassays, protein A immunoassays, to name but afew. Such assays are routine and well known in the art (see, e.g.,Ausubel et al., eds., 1994, Current Protocols in Molecular Biology, Vol.1, John Wiley & Sons, Inc., New York, which is incorporated by referenceherein in its entirety).

The binding affinity of an antibody to an influenza virus hemagglutininpolypeptide and the off-rate of an antibody-antigen interaction can bedetermined by competitive binding assays. One example of a competitivebinding assay is a radioimmunoassay comprising the incubation of labeledantigen (e.g., ³H or ¹²⁵I) with the antibody of interest in the presenceof increasing amounts of unlabeled antigen, and the detection of theantibody bound to the labeled antigen. The affinity of the antibody foran influenza virus hemagglutinin polypeptide and the binding off-ratescan be determined from the data by Scatchard plot analysis. Competitionwith a second antibody can also be determined using radioimmunoassays.In this case, an influenza virus hemagglutinin polypeptide is incubatedwith the test antibody conjugated to a labeled compound (e.g., ³H or¹²⁵I) in the presence of increasing amounts of an unlabeled secondantibody.

In certain embodiments, antibody binding affinity and rate constants aremeasured using the KinExA 3000 System (Sapidyne Instruments, Boise,Id.). In some embodiments, surface plasmon resonance (e.g., BIAcorekinetic) analysis is used to determine the binding on and off rates ofthe antibodies to an influenza virus hemagglutinin polypeptide. BIAcorekinetic analysis comprises analyzing the binding and dissociation ofinfluenza virus hemagglutinin polypeptide from chips with immobilizedantibodies to an influenza virus hemagglutinin polypeptide on theirsurface. A typical BIAcore kinetic study involves the injection of 250μL of an antibody reagent (mAb, Fab) at varying concentration in HBSbuffer containing 0.005% Tween-20 over a sensor chip surface, onto whichhas been immobilized the influenza virus hemagglutinin polypeptide. Theflow rate is maintained constant at 75 μL/min. Dissociation data iscollected for 15 min or longer as necessary. Following eachinjection/dissociation cycle, the bound antibody is removed from theinfluenza virus hemagglutinin polypeptide surface using brief, 1 minpulses of dilute acid, typically 10-100 mM HCl, though other regenerantsare employed as the circumstances warrant. More specifically, formeasurement of the rates of association, k_(on), and dissociation,k_(off), the polypeptide is directly immobilized onto the sensor chipsurface through the use of standard amine coupling chemistries, namelythe EDC/NHS method (EDC=N-diethylaminopropyl)-carbodiimide). Briefly, a5-100 nM solution of the polypeptide in 10 mM NaOAc, pH 4 or pH 5 isprepared and passed over the EDC/NHS-activated surface untilapproximately 30-50 RU's worth of polypeptide are immobilized. Followingthis, the unreacted active esters are “capped” off with an injection of1M Et-NH₂. A blank surface, containing no polypeptide, is prepared underidentical immobilization conditions for reference purposes. Once anappropriate surface has been prepared, a suitable dilution series ofeach one of the antibody reagents is prepared in HBS/Tween-20, andpassed over both the polypeptide and reference cell surfaces, which areconnected in series. The range of antibody concentrations that areprepared varies, depending on what the equilibrium binding constant,K_(D), is estimated to be. As described above, the bound antibody isremoved after each injection/dissociation cycle using an appropriateregenerant.

The neutralizing activity of an antibody can be determined utilizing anyassay known to one skilled in the art. Antibodies described herein canbe assayed for their ability to inhibit the binding of an influenzavirus, or any other composition comprising influenza virus hemagglutininpolypeptide (e.g., a VLP, liposome, or detergent extract), to its hostcell receptor (i.e., sialic acid) using techniques known to those ofskill in the art. For example, cells expressing influenza virusreceptors can be contacted with a composition comprising influenza virushemagglutinin polypeptide in the presence or absence of the antibody andthe ability of the antibody to inhibit the antigen's binding canmeasured by, for example, flow cytometry or a scintillation assay. Thecomposition comprising an influenza virus hemagglutinin polypeptide orthe antibody can be labeled with a detectable compound such as aradioactive label (e.g., ³²P, ³⁵S, and ¹²⁵I) or a fluorescent label(e.g., fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine) toenable detection of an interaction between the composition comprising aninfluenza virus hemagglutinin polypeptide and a cell receptor.Alternatively, the ability of antibodies to inhibit an influenza virushemagglutinin polypeptide from binding to its receptor can be determinedin cell-free assays. For example, a composition comprising an influenzavirus hemagglutinin polypeptide can be contacted with an antibody andthe ability of the antibody to inhibit the composition comprising aninfluenza virus hemagglutinin polypeptide from binding to a cellreceptor can be determined. In a specific embodiment, the antibody isimmobilized on a solid support and the composition comprising aninfluenza virus hemagglutinin polypeptide is labeled with a detectablecompound. Alternatively, a composition comprising an influenza virushemagglutinin polypeptide is immobilized on a solid support and theantibody is labeled with a detectable compound. In certain embodiments,the ability of an antibody to inhibit an influenza virus hemagglutininpolypeptide from binding to a cell receptor is determined by assessingthe percentage of binding inhibition of the antibody relative to acontrol (e.g., an antibody known to inhibit the influenza virushemagglutinin polypeptide from binding to the cell receptor).

In other embodiments, an antibody suitable for use in the methodsdescribed herein does not inhibit influenza virus receptor binding, yetis still found to be neutralizing in an assay described herein. In someembodiments, an antibody suitable for use in accordance with the methodsdescribed herein reduces or inhibits virus-host membrane fusion in anassay known in the art or described herein.

In one embodiment, virus-host membrane fusion is assayed in an in vitroassay using an influenza virus containing a reporter and a host cellcapable of being infected with the virus. An antibody inhibits fusion ifreporter activity is inhibited or reduced compared to a negative control(e.g., reporter activity in the presence of a control antibody or in theabsence of antibody).

In one embodiment, virus-host membrane fusion is detected using a modelsystem of cell fusion. In an exemplary cell fusion assay, cells (e.g.,HeLa cells) are transfected with a plasmid encoding an influenzahemagglutinin polypeptide and contacted and exposed to a buffer thatallows the hemagglutinin polypeptide fusion function (e.g., pH 5.0buffer) in the presence of an antibody. An antibody is neutralizing ifit reduces or inhibits syncytia formation compared to a negative control(e.g., syncytia formation in the presence of a control antibody or inthe absence of antibody).

In other embodiments, virus-host membrane fusion is assayed using an invitro liposome-based assay. In an exemplary assay, the host cellreceptor is reconstituted into liposomes containing one half of areporter. Influenza hemagglutinin polypeptide is reconstituted intoanother set of liposomes containing another half of a reporter. When thetwo liposome populations are mixed together, fusion is detected byreconstitution of the reporter, for example, an enzymatic reaction thatcan be detected colorimetrically. The antibody inhibits fusion ifreporter activity is reduced or inhibited compared to reporter activityin an assay conducted in the absence of antibody or in the presence of acontrol antibody. In certain embodiments, the ability of an antibody toinhibit fusion is determined by assessing the percentage of fusion inthe presence of the antibody relative to the percentage of fusion in thepresence a control.

5.14.3 Assays for Testing Activity of Stimulated Cells

Cells stimulated in accordance with the methods described herein may beanalyzed, for example, for integration, transcription and/or expressionof the polynucleotide or gene(s) of interest, the number of copies ofthe gene integrated, and the location of the integration. Such analysismay be carried out at any time and may be carried out by any methodsknown in the art. In other embodiments, successful stimulation of thetarget cell with an influenza hemagglutinin stem domain polypeptidedescribed herein is determined by detecting production of neutralizingantibodies against the influenza hemagglutinin stem domain polypeptideusing methods known in the art or described herein.

In certain embodiments, subjects in which the stimulated cells, e.g.,DCs, are administered can be analyzed for location of the cells,expression of a vector-delivered polynucleotide or gene encoding theinfluenza hemagglutinin stem domain polypeptide, stimulation of animmune response (e.g., production of neutralizing antibodies against theinfluenza hemagglutinin stem domain polypeptide), and/or monitored forsymptoms associated with influenza virus infection or a diseaseassociated therewith by any methods known in the art or describedherein.

Reporter assays can be used to determine the specificity of thetargeting of the influenza hemagglutinin stem domain polypeptide. Forexample, a mixed population of bone marrow cells can be obtained from asubject and cultured in vitro. The influenza hemagglutinin stem domainpolypeptide can be administered to the mixed population of bone marrowcells, and expression of a reporter gene associated with the influenzahemagglutinin stem domain polypeptide can be assayed in the culturedcells. In some embodiments, at least about 50%, more preferably at leastabout 60%, 70%, 80% or 90%, still more preferably at least about 95% ofstimulated cells in the mixed cell population are dendritic cells.

5.14.4 Antiviral Activity Assays

Antibodies described herein or compositions thereof can be assessed invitro for antiviral activity. In one embodiment, the antibodies orcompositions thereof are tested in vitro for their effect on growth ofan influenza virus. Growth of influenza virus can be assessed by anymethod known in the art or described herein (e.g. in cell culture). In aspecific embodiment, cells are infected at a MOI of 0.0005 and 0.001,0.001 and 0.01, 0.01 and 0.1, 0.1 and 1, or 1 and 10, or a MOI of0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5 or 10 and incubatedwith serum free media supplemented. Viral titers are determined in thesupernatant by hemagglutinin plaques or any other viral assay describedherein. Cells in which viral titers can be assessed include, but are notlimited to, EFK-2 cells, Vero cells, MDCK cells, primary human umbilicalvein endothelial cells (HUVEC), H292 human epithelial cell line and HeLacells. In vitro assays include those that measure altered viralreplication (as determined, e.g., by plaque formation) or the productionof viral proteins (as determined, e.g., by Western blot analysis) orviral RNAs (as determined, e.g., by RT-PCR or northern blot analysis) incultured cells in vitro using methods which are well known in the art ordescribed herein.

In one non-limiting example, a monolayer of the target mammalian cellline is infected with different amounts (e.g., multiplicity of 3 plaqueforming units (pfu) or 5 pfu) of virus (e.g., influenza) andsubsequently cultured in the presence or absence of various dilutions ofantibodies (e.g., 0.1 μg/ml, 1 μg/ml, 5 μg/ml, or 10 μg/ml). Infectedcultures are harvested 48 hours or 72 hours post infection and titeredby standard plaque assays known in the art on the appropriate targetcell line (e.g., Vero cells).

In a non-limiting example of a hemagglutination assay, cells arecontacted with an antibody and are concurrently or subsequently infectedwith the virus (e.g., at an MOI of 1) and the virus is incubated underconditions to permit virus replication (e.g., 20-24 hours). Theantibodies are preferably present throughout the course of infection.Viral replication and release of viral particles is then determined byhemagglutination assays using 0.5% chicken red blood cells. See, e.g.,Kashyap et al., PNAS USA 105: 5986-5991. In some embodiments, a compoundis considered an inhibitor of viral replication if it reduces viralreplication by at least 2 wells of HA, which equals approximately a 75%reduction in viral titer. In specific embodiments, an inhibitor reducesviral titer in this assay by 50% or more, by 55% or more, by 60% ormore, by 65% or more, by 70% or more, by 75% or more, by 80% or more, by85% or more, by 90% or more, or by 95% or more. In other specificembodiments an inhibitor results in a reduction of approximately 1 logor more, approximately 2 logs or more, approximately 3 logs or more,approximately 4 logs or more, approximately 5 logs or more,approximately 6 logs or more, approximately 7 logs or more,approximately 8 logs or more, approximately 9 logs or more,approximately 10 logs or more, 1 to 3 logs, 1 to 5 logs, 1 to 8 logs, 1to 9 logs, 2 to 10 logs, 2 to 5 logs, 2 to 7 logs, 2 logs to 8 logs, 2to 9 logs, 2 to 10 logs 3 to 5 logs, 3 to 7 logs, 3 to 8 logs, 3 to 9logs, 4 to 6 logs, 4 to 8 logs, 4 to 9 logs, 5 to 6 logs, 5 to 7 logs, 5to 8 logs, 5 to 9 logs, 6 to 7 logs, 6 to 8 logs, 6 to 9 logs, 7 to 8logs, 7 to 9 logs, or 8 to 9 logs in influenza virus titer in thesubject. The log-reduction in Influenza virus titer may be as comparedto a negative control, as compared to another treatment, or as comparedto the titer in the patient prior to antibody administration.

5.14.5 Cytotoxicity Assays

Many assays well-known in the art can be used to assess viability ofcells (infected or uninfected) or cell lines following exposure to anactive compound or a composition thereof and, thus, determine thecytotoxicity of the compound or composition. For example, cellproliferation can be assayed by measuring Bromodeoxyuridine (BrdU)incorporation (See, e.g., Hoshino et al., 1986, Int. J. Cancer 38, 369;Campana et al., 1988, J. Immunol. Meth. 107:79), (3H) thymidineincorporation (See, e.g., Chen, J., 1996, Oncogene 13:1395-403; Jeoung,J., 1995, J. Biol. Chem. 270:18367 73), by direct cell count, or bydetecting changes in transcription, translation or activity of knowngenes such as proto-oncogenes (e.g., fos, myc) or cell cycle markers(Rb, cdc2, cyclin A, D1, D2, D3, E, etc). The levels of such protein andmRNA and activity can be determined by any method well known in the art.For example, protein can be quantitated by known immunodiagnosticmethods such as ELISA, Western blotting or immunoprecipitation usingantibodies, including commercially available antibodies. mRNA can bequantitated using methods that are well known and routine in the art,for example, using northern analysis, RNase protection, or polymerasechain reaction in connection with reverse transcription. Cell viabilitycan be assessed by using trypan-blue staining or other cell death orviability markers known in the art. In a specific embodiment, the levelof cellular ATP is measured to determined cell viability.

In specific embodiments, cell viability is measured in three-day andseven-day periods using an assay standard in the art, such as theCellTiter-Glo Assay Kit (Promega) which measures levels of intracellularATP. A reduction in cellular ATP is indicative of a cytotoxic effect. Inanother specific embodiment, cell viability can be measured in theneutral red uptake assay. In other embodiments, visual observation formorphological changes may include enlargement, granularity, cells withragged edges, a filmy appearance, rounding, detachment from the surfaceof the well, or other changes. These changes are given a designation ofT (100% toxic), PVH (partially toxic-very heavy-80%), PH (partiallytoxic-heavy-60%), P (partially toxic-40%), Ps (partiallytoxic-slight-20%), or 0 (no toxicity-0%), conforming to the degree ofcytotoxicity seen. A 50% cell inhibitory (cytotoxic) concentration(IC₅₀) is determined by regression analysis of these data.

In a specific embodiment, the cells used in the cytotoxicity assay areanimal cells, including primary cells and cell lines. In someembodiments, the cells are human cells. In certain embodiments,cytotoxicity is assessed in one or more of the following cell lines:U937, a human monocyte cell line; primary peripheral blood mononuclearcells (PBMC); Huh7, a human hepatoblastoma cell line; 293T, a humanembryonic kidney cell line; and THP-1, monocytic cells. In certainembodiments, cytotoxicity is assessed in one or more of the followingcell lines: MDCK, MEF, Huh 7.5, Detroit, or human tracheobronchialepithelial (HTBE) cells.

Active compounds or compositions thereof can be tested for in vivotoxicity in animal models. For example, animal models, described hereinand/or others known in the art, used to test the activities of activecompounds can also be used to determine the in vivo toxicity of thesecompounds. For example, animals are administered a range ofconcentrations of active compounds. Subsequently, the animals aremonitored over time for lethality, weight loss or failure to gainweight, and/or levels of serum markers that may be indicative of tissuedamage (e.g., creatine phosphokinase level as an indicator of generaltissue damage, level of glutamic oxalic acid transaminase or pyruvicacid transaminase as indicators for possible liver damage). These invivo assays may also be adapted to test the toxicity of variousadministration mode and/or regimen in addition to dosages.

The toxicity and/or efficacy of an active compound can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀. Anactive compound that exhibits large therapeutic indices is preferred.While an active compound that exhibits toxic side effects may be used,care should be taken to design a delivery system that targets suchagents to the site of affected tissue in order to minimize potentialdamage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage of an active compound for use inhumans. The dosage of such agents lies preferably within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anyactive compound used in a method described herein, the effective dosecan be estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound that achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high-performance liquid chromatography.Additional information concerning dosage determination is providedherein.

Further, any assays known to those skilled in the art can be used toevaluate the prophylactic and/or therapeutic utility of the activecompounds and compositions described herein, for example, by measuringviral infection or a condition or symptoms associated therewith.

5.14.6 In Vivo Antiviral Activity

Active compounds and compositions thereof are preferably assayed in vivofor the desired therapeutic or prophylactic activity prior to use inhumans. For example, in vivo assays can be used to determine whether itis preferable to administer an active compound or composition thereofand/or another therapy. For example, to assess the use of an activecompound or composition thereof to prevent an influenza virus disease,the composition can be administered before the animal is infected withinfluenza virus. Alternatively, or in addition, an active compound orcomposition thereof can be administered to the animal at the same timethat the animal is infected with influenza virus. To assess the use ofan active compound or composition thereof to treat an influenza virusinfection or disease associated therewith, the compound or compositionmay be administered after infecting the animal with influenza virus. Ina specific embodiment, an active compound or composition thereof isadministered to the animal more than one time.

Active compounds and compositions thereof can be tested for antiviralactivity in animal model systems including, but are not limited to,rats, mice, chicken, cows, monkeys, pigs, goats, sheep, dogs, rabbits,guinea pigs, etc. In a specific embodiment, active compounds andcompositions thereof are tested in a mouse model system. Such modelsystems are widely used and well-known to the skilled artisan. In aspecific embodiment, active compounds and compositions thereof aretested in a mouse model system. Non-limiting examples of animal modelsfor influenza virus are provided in this section.

In general, animals are infected with influenza virus and concurrentlyor subsequently treated with an active compound or composition thereof,or placebo. Alternatively, animals are treated with an active compoundor composition thereof or placebo and subsequently infected withinfluenza virus. Samples obtained from these animals (e.g., serum,urine, sputum, semen, saliva, plasma, or tissue sample) can be testedfor viral replication via well known methods in the art, e.g., thosethat measure altered viral titers (as determined, e.g., by plaqueformation), the production of viral proteins (as determined, e.g., byWestern blot, ELISA, or flow cytometry analysis) or the production ofviral nucleic acids (as determined, e.g., by RT-PCR or northern blotanalysis). For quantitation of virus in tissue samples, tissue samplesare homogenized in phosphate-buffered saline (PBS), and dilutions ofclarified homogenates are adsorbed for 1 hour at 37° C. onto monolayersof cells (e.g., Vero, CEF or MDCK cells). In other assays,histopathologic evaluations are performed after infection, preferablyevaluations of the organ(s) the virus is known to target for infection.Virus immunohistochemistry can be performed using a viral-specificmonoclonal antibody.

The effect of an active compound or composition thereof on the virulenceof a virus can also be determined using in vivo assays in which thetiter of the virus in an infected subject administered an activecompound or composition thereof, the length of survival of an infectedsubject administered an active compound or composition thereof, theimmune response in an infected subject administered an active compoundor composition thereof, the number, duration and/or severity of thesymptoms in an infected subject administered an active compound orcomposition thereof, and/or the time period before onset of one or moresymptoms in an infected subject administered an active compound orcomposition thereof, is assessed. Techniques known to one of skill inthe art can be used to measure such effects. In certain embodiments, anactive compound or composition thereof results in a 0.5 fold, 1 fold, 2fold, 4 fold, 6 fold, 8 fold, 10 fold, 15 fold, 20 fold, 25 fold, 50fold, 75 fold, 100 fold, 125 fold, 150 fold, 175 fold, 200 fold, 300fold, 400 fold, 500 fold, 750 fold, or 1,000 fold or greater reductionin titer of influenza virus relative to an untreated subject. In someembodiments, an active compound or composition thereof results in areduction in titer of influenza virus relative to an untreated subjectof approximately 1 log or more, approximately 2 logs or more,approximately 3 logs or more, approximately 4 logs or more,approximately 5 logs or more, approximately 6 logs or more,approximately 7 logs or more, approximately 8 logs or more,approximately 9 logs or more, approximately 10 logs or more, 1 to 3logs, 1 to 5 logs, 1 to 8 logs, 1 to 9 logs, 2 to 10 logs, 2 to 5 logs,2 to 7 logs, 2 logs to 8 logs, 2 to 9 logs, 2 to 10 logs 3 to 5 logs, 3to 7 logs, 3 to 8 logs, 3 to 9 logs, 4 to 6 logs, 4 to 8 logs, 4 to 9logs, 5 to 6 logs, 5 to 7 logs, 5 to 8 logs, 5 to 9 logs, 6 to 7 logs, 6to 8 logs, 6 to 9 logs, 7 to 8 logs, 7 to 9 logs, or 8 to 9 logs.

Influenza virus animal models, such as ferret, mouse, guinea pig,squirrel monkey, macaque, and chicken, developed for use to testantiviral agents against influenza virus have been described. See, e.g.,Sidwell et al., Antiviral Res., 2000, 48:1-16; Lowen A. C. et al. PNAS.,2006, 103: 9988-92; and McCauley et al., Antiviral Res., 1995,27:179-186 and Rimmelzwann et al., Avian Diseases, 2003, 47:931-933. Formouse models of influenza, non-limiting examples of parameters that canbe used to assay antiviral activity of active compounds administered tothe influenza-infected mice include pneumonia-associated death, serumal-acid glycoprotein increase, animal weight, lung virus assayed byhemagglutinin, lung virus assayed by plaque assays, andhistopathological change in the lung. Statistical analysis is carriedout to calculate significance (e.g., a P value of 0.05 or less).

In other assays, histopathologic evaluations are performed afterinfection of an animal model subject. Nasal turbinates and trachea maybe examined for epithelial changes and subepithelial inflammation. Thelungs may be examined for bronchiolar epithelial changes andperibronchiolar inflammation in large, medium, and small or terminalbronchioles. The alveoli are also evaluated for inflammatory changes.The medium bronchioles are graded on a scale of 0 to 3+ as follows: 0(normal: lined by medium to tall columnar epithelial cells with ciliatedapical borders and basal pseudostratified nuclei; minimal inflammation);1+(epithelial layer columnar and even in outline with only slightlyincreased proliferation; cilia still visible on many cells);2+(prominent changes in the epithelial layer ranging from attenuation tomarked proliferation; cells disorganized and layer outline irregular atthe luminal border); 3+(epithelial layer markedly disrupted anddisorganized with necrotic cells visible in the lumen; some bronchiolesattenuated and others in marked reactive proliferation).

The trachea is graded on a scale of 0 to 2.5+ as follows: 0 (normal:Lined by medium to tall columnar epithelial cells with ciliated apicalborder, nuclei basal and pseudostratified. Cytoplasm evident betweenapical border and nucleus. Occasional small focus with squamous cells);1+ (focal squamous metaplasia of the epithelial layer); 2+ (diffusesquamous metaplasia of much of the epithelial layer, cilia may beevident focally); 2.5+ (diffuse squamous metaplasia with very few ciliaevident).

Virus immunohistochemistry is performed using a viral-specificmonoclonal antibody (e.g. NP-, N- or HN-specific monoclonal antibodies).Staining is graded 0 to 3+ as follows: 0 (no infected cells); 0.5+ (fewinfected cells); 1+ (few infected cells, as widely separated individualcells); 1.5+ (few infected cells, as widely separated singles and insmall clusters); 2+ (moderate numbers of infected cells, usuallyaffecting clusters of adjacent cells in portions of the epithelial layerlining bronchioles, or in small sublobular foci in alveoli); 3+(numerous infected cells, affecting most of the epithelial layer inbronchioles, or widespread in large sublobular foci in alveoli).

In one example, the ability to induce lung lesions and cause infectionin an animal model of virus infection is compared using wild-type virusand mock virus. Lung lesions can be assessed as a percentage of lunglobes that are healthy by visual inspection. Animals are euthanized 5days p.i. by intravenous administration of pentobarbital, and theirlungs are removed in toto. The percentage of the surface of eachpulmonary lobe that is affected by macroscopic lesions is estimatedvisually. The percentages are averaged to obtain a mean value for the 7pulmonary lobes of each animal. In other assays, nasal swabs can betested to determine virus burden or titer. Nasal swabs can be takenduring necropsy to determine viral burden post-infection.

In one embodiment, virus is quantified in tissue samples. For example,tissue samples are homogenized in phosphate-buffered saline (PBS), anddilutions of clarified homogenates adsorbed for 1 h at 37° C. ontomonolayers of cells (e.g., MDCK cells). Infected monolayers are thenoverlaid with a solution of minimal essential medium containing 0.1%bovine serum albumin (BSA), 0.01% DEAE-dextran, 0.1% NaHCO₃, and 1%agar. Plates are incubated 2 to 3 days until plaques could bevisualized. Tissue culture infectious dose (TCID) assays to titratevirus from PR8-infected samples are carried out as follows. Confluentmonolayers of cells (e.g., MDCK cells) in 96-well plates are incubatedwith log dilutions of clarified tissue homogenates in media. Two tothree days after inoculation, 0.05-ml aliquots from each well areassessed for viral growth by hemagglutination assay (HA assay).

5.14.6.1.1 Assays in Humans

In one embodiment, an active compound or composition thereof thatmodulates replication an influenza virus are assessed in infected humansubjects. In accordance with this embodiment, an active compound orcomposition thereof is administered to the human subject, and the effectof the active compound or composition on viral replication is determinedby, e.g., analyzing the level of the virus or viral nucleic acids in abiological sample (e.g., serum or plasma). An active compound orcomposition thereof that alters virus replication can be identified bycomparing the level of virus replication in a subject or group ofsubjects treated with a control to that in a subject or group ofsubjects treated with an active compound or composition thereof.Alternatively, alterations in viral replication can be identified bycomparing the level of the virus replication in a subject or group ofsubjects before and after the administration of an active compound orcomposition thereof. Techniques known to those of skill in the art canbe used to obtain the biological sample and analyze the mRNA or proteinexpression.

In another embodiment, the effect of an active compound or compositionthereof on the severity of one or more symptoms associated with aninfluenza virus infection/disease are assessed in an infected subject.In accordance with this embodiment, an active compound or compositionthereof or a control is administered to a human subject suffering frominfluenza virus infection and the effect of the active compound orcomposition on one or more symptoms of the virus infection isdetermined. An active compound or composition thereof that reduces oneor more symptoms can be identified by comparing the subjects treatedwith a control to the subjects treated with the active compound orcomposition. In another embodiment, an active compound or compositionthereof is administered to a healthy human subject and monitored forefficacy as a vaccine (e.g., the subject is monitored for the onset ofsymptoms of influenza virus infection; the ability of influenza virus toinfect the subject; and/or a reduction in/absence of one or moresymptoms associated with influenza virus infection). Techniques known tophysicians familiar with infectious diseases can be used to determinewhether an active compound or composition thereof reduces one or moresymptoms associated with the influenza virus disease.

5.15 Kits

Provided herein is a pharmaceutical pack or kit comprising one or morecontainers filled with one or more of the ingredients of thepharmaceutical compositions described herein, such as one or more activecompounds provided herein. Optionally associated with such container(s)can be a notice in the form prescribed by a governmental agencyregulating the manufacture, use or sale of pharmaceuticals or biologicalproducts, which notice reflects approval by the agency of manufacture,use or sale for human administration.

The kits encompassed herein can be used in the above methods. In oneembodiment, a kit comprises an active compound described herein,preferably one or more influenza hemagglutinin stem domain polypeptides,in one or more containers. In certain embodiments, a kit comprises avaccine described herein, e.g., a split virus vaccine, a subunitvaccine, an inactivated influenza virus vaccine, or a live influenzavirus vaccine.

6. EXAMPLES 6.1 Example 1: Influenza Hemagglutinin Stem DomainPolypeptides

This example describes the generation of constructs that expressinfluenza hemagglutinin stem domain polypeptides. The influenzahemagglutinin stem domain polypeptides lack the globular head domain ofinfluenza virus hemagglutinin and maintain the structural integrity ofthe stalk region of the influenza virus hemagglutinin. Since the stalkregion of influenza virus hemagglutinin is relatively conserved amonginfluenza viruses, the influenza hemagglutinin stem domain polypeptidesshould induce neutralizing antibodies against the stalk region ofhemagglutinin that are cross-reactive with influenza virus hemagglutininfrom different influenza virus subtypes and strains.

FIG. 3 depicts two schematic nucleotide constructs for expressing aninfluenza HA stem domain polypeptide from influenza A HK68-H3N2. FIG. 3also depicts a schematic of a construct (WT HA) for expressing fulllength influenza HA. The first construct (“Membrane Bound HA”) providesa nucleotide sequence encoding the N-terminal and C-terminal segments,linker peptides, and an HA2 domain. The first construct also encodes atransmembrane (TM) domain and a cytoplasmic (CT) domain. The firstconstruct also encodes a signal peptide (SP).

Additionally, a second construct (“Soluble HA”) provides a nucleotidesequence not encoding the SP, TM or CT in order to generate soluble formof the influenza HA stem domain polypeptide. The second constructincludes, after the sequence encoding the HA2 domain, a nucleotidesequence encoding a thrombin cleavage site, a trimerization domain, anda His-tag.

FIG. 4 illustrates the location of the linker peptide in a putativethree dimensional structure of an influenza HA stem domain polypeptide.

Constructs:

Construct #1:

The nucleotide sequence encoding amino acids 53 to 276 of the influenzaHA1 domain was deleted from the full-length influenza virus A/PuertoRico/8/34 (PR8; H1N1) hemagglutinin and replaced by a linker sequenceencoding two glycine residues (GG). FIG. 6 provides the PR8 HA construct(PR8 HAΔGHD (2G)) with a GG linker with both nucleotide (SEQ ID NO:169)and amino acid (SEQ ID NO:170) sequences. A similar construct was madeusing the full-length influenza virus A/Hong Kong/1/68 (HK68; H3N2)hemagglutinin polypeptide (HK68 HAΔGHD (2G)) and the construct wasinserted in the pCAGGS expression vector. The PR8 HAΔGHD (2G) and HK68HAΔGHD (2G) constructs were each inserted into a pPol1 vector for use inthe rescue of recombinant influenza virus.

Construct #2:

The nucleotide sequence encoding amino acids 53 to 276 of the influenzaHA1 domain was deleted from the full-length PR8 hemagglutinin andreplaced by a linker sequence encoding four glycine residues (GGGG; SEQID NO:319). FIG. 7 provides the PR8 HA construct (PR8 HAΔGHD (4G)) witha GGGG (SEQ ID NO:319) linker with both nucleotide (SEQ ID NO:171) andamino acid (SEQ ID NO:172) sequences. A similar construct was made usingthe full-length influenza virus HK68, H3N2 hemagglutinin polypeptide(HK68 HAΔGHD (4G)). The PR8 HAΔGHD (4G) and HK68 HAΔGHD (4G) constructswere each inserted in the pCAGGS expression vector. The PR8 HAΔGHD (4G)and HK68 HAΔGHD (4G) constructs were each inserted into a pPol1 vectorfor use in the rescue of recombinant influenza virus.

Construct #3:

The nucleotide sequence encoding amino acids 53 to 276 of the influenzaHA1 domain was deleted from the full-length PR8 hemagglutinin andreplaced by a linker sequence encoding a proline residue immediatelyfollowed by a glycine residue (PG). FIG. 8 provides the PR8 HA construct(PR8 HAΔGHD (PG)) with a PG linker with both nucleotide (SEQ ID NO:173)and amino acid (SEQ ID NO:174) sequences. A similar construct was madeusing the full-length influenza virus HK68, H3N2 hemagglutininpolypeptide (HK68 HAΔGHD (PG)). The PR8 HAΔGHD (PG) and HK68 HAΔGHD (PG)constructs were each inserted in the pCAGGS expression vector. The PR8HAΔGHD (PG) and HK68 HAΔGHD (PG) constructs were each inserted into apPol1 vector for use in the rescue of recombinant influenza virus.

Construct #4:

The nucleotide sequence encoding amino acids 53 to 276 of the influenzaHA1 domain is deleted from the full-length PR8 and replaced by a linkersequence encoding four glycine residues (GGGG; SEQ ID NO:319). FIG. 9provides the PR8 HA construct with a GGGG (SEQ ID NO:319) linker withboth nucleotide (SEQ ID NO:175) and amino acid (SEQ ID NO:176)sequences. The PR8 HA construct in FIG. 9 also encodes, after theinfluenza HA2 domain, a thrombin cleavage site, a foldon domain fortrimerization and a HIS₆ tag. A similar construct may be made using theinfluenza virus HK68, H3N2 hemagglutinin polypeptide.

Expression of Constructs:

The pCAGGS expression vectors containing either the HK68 HAΔGHD (2G)construct, PR8 HAΔGHD (4G) construct, HK68 HAΔGHD (4G) construct, PR8HAΔGHD (PG) construct, or HK68 HAΔGHD (PG) construct were transientlytransfected into 293T cells in the absence of exogenous trypsin.Influenza HA stem domain polypeptides HAΔGHD were shown to be expressedin human 293T cell cultures. At 24 hours post-transfection, cells werelysed and lysates were subjected to SDS-PAGE followed by Westernblotting. Either a rabbit polyclonal antiserum raised against a HK68influenza A virus HA2 preparation or a mouse monoclonal raised againstmultiple H3 HA proteins was used as a primary antibody, as indicated atthe bottom of each blot shown in FIGS. 5A and 5B.

As shown in FIG. 5A, polyclonal antibodies against a HK68 influenza Avirus HA2 preparation recognized full length HA0 expressed by thecontrol construct (lane 2) and truncated HA0 (HA0ΔGHD) expressed by thePR8 HAΔGHD (4G) construct (lane 3) and PR8 HAΔGHD (PG) construct (lane4).

As shown in FIG. 5B, monoclonal antibodies against multiple H3 HAproteins also recognized full length HA0 expressed by the controlconstruct (lane 2) and truncated HA0 (HA0ΔGHD) expressed by the HK68HAΔGHD (2G) construct (lane 3), HK68 HAΔGHD (4G) construct (lane 4) andHK68 HAΔGHD (PG) construct (lane 5).

6.2 Example 2: Influenza Virus Vaccine Based on Conserved HemagglutininStalk Domain

This example describes the effectiveness of an influenza hemagglutininstem domain polypeptide (sometimes referred to herein as a “headlessHA”) vaccine in inducing an immune response that provides fullprotection against death and partial protection against diseasefollowing lethal viral challenge.

6.2.1 Materials and Methods

Plasmids

pGag-EGFP was generously provided by Carol Carter, Stonybrook University(Hermida-Matsumoto, L., and M. D. Resh. 2000. Localization of humanimmunodeficiency virus type 1 Gag and Env at the plasma membrane byconfocal imaging. J Virol 74:8670-9). The pCAGGS expression plasmid waskindly provided by J. Miyazaki, Osaka University (Miyazaki, J., S.Takaki, K. Araki, F. Tashiro, A. Tominaga, K. Takatsu, and K. Yamamura.1989. Expression vector system based on the chicken beta-actin promoterdirects efficient production of interleukin-5. Gene 79:269-77). Theplasmids pDZ PR8 HA and pDZ PR8 NA were constructed previously asdescribed in reference (Quinlivan, M., D. Zamarin, A. Garcia-Sastre, A.Cullinane, T. Chambers, and P. Palese. 2005. Attenuation of equineinfluenza viruses through truncations of the NS1 protein. J Virol79:8431-9). For the construction of pCAGGS HK68 HA and pCAGGS HK68 NA,viral genes were reverse transcribed (Transcriptor RT, Roche) frompurified virion RNA, amplified (PFU turbo, Stratagene) and cloned intothe vector pPOL1 (Fodor, E., L. Devenish, O. G. Engelhardt, P. Palese,G. G. Brownlee, and A. Garcia-Sastre. 1999. Rescue of influenza A virusfrom recombinant DNA. J Virol 73:9679-82) following the recombinationalprotocol described by Wang et al. (Wang, S., Q. Liu, J. Pu, Y. Li, L.Keleta, Y. W. Hu, J. Liu, and E. G. Brown. 2008. Simplifiedrecombinational approach for influenza A virus reverse genetics. J VirolMethods 151:74-8). Protein coding regions were then amplified withprimers carrying the appropriate restriction enzyme sites and subclonedinto the multiple cloning site of pCAGGS between the Not1 and Nhe1sites. Headless HA constructs were generated by either excise or fusionPCR methods. Excise PCR was performed on the pPOL1 PR8 HA or pPOL1 HK68HA plasmids. The resulting PCR products were circularized by ligationand the open reading frame of the headless HA was then subcloned intopCAGGS at the Not1 and Nhe1 sites. Fusion PCR was performed on pDZ PR8HA or pCAGGS HK68 plasmid templates and products were inserted intopCAGGS at the Not1 and Nru1 sites. Primer sequences used are as followsTable 5 below.

TABLE 5Summary of Sequences of Primers Used in the Construction of HeadlessHAs Listed in FIG. 10 PCR Construct method^(a) Upstream primer^(b)Downstream primer^(c) PR8 2G Excise ACATAGTTTTCCGTTGTGGCTGTCTTCGAGCGGAGGCTGTAACACGAAGTGTCAAACACCCCTGGGAGCTATA [SEQ ID NO: 274] AACA[SEQ ID NO: 291] PR8 4G Excise ACATAGTTTTCCGTTGTGGCTGTCTTCGAGCGGAGGCGGAGGTTGTAACACGAAGTGTCAAACACCCCTGGGA [SEQ ID NO: 275] GCTATAAACA[SEQ ID NO: 292] PR8 PG Excise ACATAGTTTTCCGTTGTGGCTGTCTTCGAGCCCAGGCTGTAACACGAAGTGTCAAACACCCCTGGGAGCTATA [SEQ ID NO: 276] AACA[SEQ ID NO: 293] PR8 No Fusion TGACACTTCGTGTTACCTAGTTTTCCGTTGTGGCTGGGTAACACGAAGTGTCAAACAC Cys 1G [SEQ ID NO: 277] [SEQ ID NO: 294] PR8 NoFusion ACTTCGTGTTTCCGCCTAGTTTTCCGTTGTGGCTG GGCGGAAACACGAAGTGTCAAACACCys 2G [SEQ ID NO: 278] [SEQ ID NO: 295] PR8 No FusionCGTGTTACCTCCGCCTAGTTTTCCGTTGTGGCTG GGCGGAGGTAACACGAAGTGTCAAACAC Cys 3G[SEQ ID NO: 279] [SEQ ID NO: 296] PR8 No FusionTGTTTGACACTTCGTGTTTAGTTTTCCGTTGTGGCTGTCGCCACAACGGAAAACTAAACACGAAGTGTCAAACACCC Cys [SEQ ID NO: 280][SEQ ID NO: 297] PR8 No Fusion GGTGTTTGACACTTCGTTAGTTTTCCGTTGTGGCTGTCGCCACAACGGAAAACTAACGAAGTGTCAAACACCCCTG Cys Δ1 [SEQ ID NO: 281][SEQ ID NO: 298] PR8 No Fusion AGGGGTGTTTGACACTTTTTTCCGTTGTGGCTGTCTTCACAGCCACAACGGAAAAAAGTGTCAAACACCCCTGGGA Cys Δ3 [SEQ ID NO: 282][SEQ ID NO: 299] PR8 No Fusion ACTTCGTGTTGGAGGCGTTTAGTTTTCCGTTGTGGCTGAACGCCTCCAACACGAAGTGTCAAACAC Cys NAS [SEQ ID NO: 283] [SEQ ID NO: 300]HK68 Excise GCUCCUCAACGGGGAAAAUAUGCGGA GGCTGTATTTCTGAATGCATCACTCC 2G[SEQ ID NO: 284] [SEQ ID NO: 301] HK68 ExciseGCUCCUCAACGGGGAAAAUAUGCGGAGGC GGAGGTTGTATTTCTGAATGCATCACTCC 4G[SEQ ID NO: 285] [SEQ ID NO: 302] HK68 Excise GCUCCUCAACGGGGAAAAUAUGCCCAGGCTGTATTTCTGAATGCATCACTCC PG [SEQ ID NO: 286] [SEQ ID NO: 303] HK68Fusion GAGTGATGCATTCAGAAATTATTTTCCCCGTTGAGGAGCCCTCAACGGGGAAAATAATTTCTGAATGCATCACTCCA No Cys [SEQ ID NO: 287][SEQ ID NO: 304] HK68 Fusion TGGAGTGATGCATTCAGATATTTTCCCCGTTGAGGAGCCCTCAACGGGGAAAATATCTGAATGCATCACTCCAAAT No Cys [SEQ ID NO: 288][SEQ ID NO: 305] Δ1 HK68 Fusion ATTTGGAGTGATGCATTCTTTCCCCGTTGAGGAGCTCCTCCTCAACGGGGAAAGAATGCATCACTCCAAATGG No Cys [SEQ ID NO: 289][SEQ ID NO: 306] Δ3 HK68 Fusion TTCAGAAATGGAGGCGTTTATTTTCCCCGTTGAGGAGAACGCCTCCATTTCTGAATGCATCACTCC No Cys [SEQ ID NO: 290] [SEQ ID NO: 307]NAS ^(a)In the Excise PCR method, primers flanking the sequence targetedfor deletion are used to amplify the remainder of the gene and plasmidvector. Thus, the downstream primer is the forward primer in the PCRreaction and the upstream primer is the reverse primer. A linearfragment is produced which lacks the intervening sequence. A shortforeign sequence can be introduced into the deletion site by adding itto the 5′ end of one of the primers. Once generated, the PCR product ispurified and self-ligated in order to produce a circular plasmidcarrying the modified gene of interest. In the Fusion PCR method twofragments of the desired product are PCR amplified independently. Onefragment corresponds to the sequence upstream of the introduced mutationand the second corresponds to the sequence downstream of the mutation.The upstream fragment is amplified using the upstream primer incombination with a 5′ outside primer, while the downstream fragment isproduced using the downstream primer together with a 3′ outside primer.The nucleotide sequence to be introduced at the site of mutation isadded to both fragments through inclusion in both the upstream anddownstream primers. The two pieces are then fused in a subsequent PCRreaction using only the 5′ and 3′ outside primers. The PR8 outsideprimers used for all PR8 fusion PCRs were PR8 5′:cgagctcatgaaggcaaacctactgg and PR8 3′: cgcagatcttcagatgcatattctgcact.The HK68 outside primers used for all HK68 fusion PCRs were HK68 5′not1:ggaagcggccgcatgaagaccatcattgctttgag and HK68 3′nru1:gcggcgtcgcgatcaaatgcaaatgttgcacctaa. ^(b)These primers annealimmediately upstream of the mutation site in both Excise and Fusion PCRmethods. ^(c)These primers anneal immediately downstream of the mutationsite in both Excise and Fusion PCR method

Antibodies

Monoclonal antibody (mAb) 12D1 was generated by sequential intramuscularimmunization of Balb/C mice with plasmid DNAs encoding the HK68 HA, theA/Alabama/1/1981 (H3N2) HA, and the A/Beijing/46/1992 (H3N2) HA,followed by a boost with whole A/Wyoming/03/2003 (H3N2) virus. See U.S.provisional application Ser. Nos. 61/181,263 and 61/224,302, filed May26, 2009 and Jul. 9, 2009, which are incorporated herein by reference intheir entirety, for a description of the 12D1 mAb. This mAb bindsmultiple H3 HA proteins and maps to the HA2 subunit. Rabbit polyclonalserum 3951 was raised against PR8 virus from which the HA1 subunit hadbeen removed by treatment with acid and DTT (Graves, P. N., J. L.Schulman, J. F. Young, and P. Palese. 1983. Preparation of influenzavirus subviral particles lacking the HA1 subunit of hemagglutinin:unmasking of cross-reactive HA2 determinants. Virology 126:106-16).

Cells and Viruses

293T cells were obtained from the ATCC and were maintained in Dulbecco'smodified Eagles medium (DMEM; Gibco) supplemented with 10% fetal bovineserum (FBS; Clontech).

Influenza A/Puerto Rico/8/1934 (H1N1) virus was obtained by reversegenetics as previously described (Steel, J., S. V. Burmakina, C. Thomas,E. Spackman, A. Garcia-Sastre, D. E. Swayne, and P. Palese. 2008. Acombination in-ovo vaccine for avian influenza virus and Newcastledisease virus. Vaccine 26:522-31) using plasmids encoding the eightgenes defined by accession numbers AF389115 to AF189122 (A/PuertoRico/8/34/Mount Sinai) in the NCBI database. The virus was amplified in10-11 day old embryonated chickens eggs and titrated by plaque assay.

Western Blotting

To assess expression levels of HA-based proteins, 293T cells weretransfected with 2 μg of the appropriate plasmid using Lipofectamine2000 (Invitrogen) according to the manufacturer's instructions. At 24 hpost-transfection, cells were lysed in 2× protein loading buffer (125 mMTris-HCl [pH 6.8], 4% sodium dodecyl sulfate, 20% glycerol, 10%β-mercaptoethanol, and 0.1% bromophenol blue). Lysates were heated at100° C. for 5 minutes and then separated on a 10% sodium dodecylsulfate-polyacrylamide gel and transferred to a nitrocellulose membrane(Whatman, Inc.). To detect HA-based proteins in VLP preparations,pelleted VLPs were suspended in phosphate buffered saline (PBS), lysedthrough 1:1 mixing with 2× protein loading buffer, boiled for 5 minutes,separated on a 10% sodium dodecyl sulfate-polyacrylamide gel andtransferred to a nitrocellulose membrane. Membranes were then probedwith mAb 12D1 or 3951 antiserum at a 1:2000 dilutions.

Flow Cytometric Analysis

To assess levels of HA-based proteins at the cell surface, 293T cellswere transfected with 1 μg of the appropriate plasmid usingLipofectamine 2000 (Invitrogen) according to the manufacturer'sinstructions. At 24 hours post-transfection, cells were trypsinized andresuspended in PBS containing 2% FBS prior to staining with 3951antiserum at a 1/250 dilution or mAb 12D1 at a 1/200 dilution. Stainedcells were enumerated on a Beckman Coulter Cytomics FC 500 flowcytometer and results were analyzed using Flow Jo software.

Generation of Virus Like Particles

For the production of virus like particles, 6×10⁶ 293T cells were seededinto a 10 cm dish in 8 ml of DMEM with 10% FBS. While still insuspension, cells were transfected with Lipofectamine 2000 (Invitrogen)combined with the desired plasmid DNAs at a 4:1 ratio and as per themanufacturer's instructions. The amounts of plasmid DNA used were asfollows: for Gag-only VLPs, 7.5 μg pGagEGFP was transfected; for gag+PR84G VLPs, 4.5 μg of pGagEGFP plus 4.5 μg of pCAGGS PR8 4G was used; forGag+HK68 4G VLPs, 4.5 μg of pGagEGFP plus 4.5 μg of pCAGGS HK68 4G wasused; and for Gag+PR8 HA+PR8 NA VLPs, 3 μg of pGagEGFP was combined with3 μg each of pDZ PR8 HA and pDZ PR8 NA. At 6 hours post-transfection,medium was changed to fresh DMEM containing 10% FBS or to Opti-MEM(Gibco) supplemented with 3% bovine serum albumin (Sigma) and 10 μg/mlTPCK-treated trypsin (Sigma). VLPs were harvested at 28 hourspost-transfection by layering clarified cell culture supernatant over acushion of 30% sucrose in NTE buffer (1M sodium chloride, 0.1 M Tris,0.01 M EDTA, pH 7.4) and centrifuging for 2 h at 4° C. and 25,000 RPM inan SW28 rotor (Beckman). Pellets were resuspended in PBS and HA proteincontent was assessed by Western blotting in parallel with serialdilutions of a known amount of gradient purified PR8 or HK68 virus(prepared as described in Palese, P., and J. L. Schulman. 1976.Differences in RNA patterns of influenza A viruses. J Virol 17:876-84).VLPs shown in FIG. 13 were produced in the presence of exogenoustrypsin, while those used to boost mice were produced without theaddition of trypsin.

Mouse Vaccine-Challenge Experiment

Female, 6 to 8-week-old C57BL/6 mice (Charles River Laboratories) wereinitially vaccinated by intramuscular administration of plasmid DNAfollowed immediately by the application of electrical stimulation(TriGrid Delivery System, Ichor Medical Systems (Luxembourg, A., C. F.Evans, and D. Hannaman. 2007. Electroporation-based DNA immunisation:translation to the clinic. Expert Opin Biol Ther 7:1647-64; Luxembourg,A., D. Hannaman, E. Nolan, B. Ellefsen, G. Nakamura, L. Chau, O. Tellez,S. Little, and R. Bernard. 2008. Potentiation of an anthrax DNA vaccinewith electroporation. Vaccine 26:5216-22)). The spacing of the TriGridelectrode array is 2.5 mm, and the electrical field is applied at anamplitude of 250 V/cm of electrode spacing for six pulses totaling 40msec duration applied over a 400 msec interval. Each DNA vaccinationcomprised 37.5 μg of pGagEGFP alone or in combination with 75 μg of pDZPR8 HA, pCAGGS PR8 4G or pCAGGS HK68 4G. Three weeks later a DNA boostwas performed following the same procedure. Five weeks after the seconddose of DNA was administered, the mice were boosted a second time withVLPs. For the HA-containing VLPs, HA content was normalized such thateach mouse received approximately 150 ng HA protein. Prior tointraperitoneal administration, VLP suspensions were combined in a 1:1ratio with complete Freund's adjuvant (Pierce) and emulsified bymultiple passes through two linked syringes. Mice were challenged threeweeks following the VLP boost via intranasal inoculation of 2 MLD50 (50%mouse lethal dose) of PR8 virus in a total volume of 50 μl PBS. Bodyweight was monitored daily and mice losing greater than 25% of theirinitial weight were sacrificed and scored as dead.

Serological Assays

Sera were collected from mice immediately prior to challenge and at 21days post-challenge. To remove non-specific inhibitors ofhemagglutination, trypsin-heat-periodate treatment was performed asdescribed previously (Lowen, A. C., J. Steel, S. Mubareka, E. Carnero,A. Garcia-Sastre, and P. Palese. 2009. Blocking inter-host transmissionof influenza virus by vaccination in the guinea pig model. J Virol. 83:2803-18). For hemagglutination inhibition assays, sera from eachvaccination group were pooled; for ELISA, sera collected from individualmice were evaluated separately. HI assays were carried out as describedpreviously (Lowen, A. C., J. Steel, S. Mubareka, E. Carnero, A.Garcia-Sastre, and P. Palese. 2009. Blocking inter-host transmission ofinfluenza virus by vaccination in the guinea pig model. J Virol. 83:2803-18). For ELISA, 96-well plates (Co-Star) were coated with 0.25 μgper well of PR8 virus or with 0.1 μg per well of purified recombinant HAprotein in PBS. PR8 virus was prepared from allantoic fluid byconcentration through a 30% sucrose cushion as previously described(Lowen, A. C., J. Steel, S. Mubareka, E. Carnero, A. Garcia-Sastre, andP. Palese. 2009. Blocking inter-host transmission of influenza virus byvaccination in the guinea pig model. J Virol. 83: 2803-18). Therecombinant HA proteins of the A/California/04/2009, A/VietNam/1203/2004, and A/Singapore/1/1957 viruses were obtained from BEIResources; the A/Hong Kong/1/1968 HA was the generous gift of IanWilson; and the A/New Caledonia/20/99 HA was purchased from Feldan-Bio.Five-fold serial dilutions of anti-sera were incubated on the platesand, after extensive washing, bound antibody was detected with analkaline phosphatase linked anti-mouse IgG antibody (Caltag) and PNPPsubstrate (Sigma). In each assay a rabbit immune serum raised againstwhole PR8 virus was included as a positive control, and serum obtainedfrom a naïve C57BL/6 mouse was included as a negative control.

6.2.2 Results

Design and Construction of Headless HA Constructs

The goal was to generate an immunogen consisting of the complete HA2polypeptide plus the regions of HA1 contributing to the stalk region,but lacking the globular head domain of HA1. With this aim in mind, theexistence of a conserved disulfide bond linking cysteines 52 and 277 (H3numbering) of HA1 was noted. The loop flanked by these two cysteinescomprises the bulk of the globular head domain, while the N-terminal 51and the C-terminal 52 amino acids of HA1 extend downward from thecysteine bridge and contribute to the stalk region. Due to the proximityof cysteines 52 and 277 in the three-dimensional structure of HA(Stevens, J., A. L. Corper, C. F. Basler, J. K. Taubenberger, P. Palese,and I. A. Wilson. 2004. Structure of the uncleaved human H1hemagglutinin from the extinct 1918 influenza virus. Science 303:1866-70and FIG. 10), it was predicted that replacement of the intervening loopwith a short linker peptide would not disrupt the folding of theremainder of the molecule. Based on this principle, a panel of headlessHA constructs was designed (FIG. 10).

First, sequences encoding linker peptides of two glycines (2G), fourglycines (4G) or a proline and a glycine (PG) were inserted into theopen reading frames of the A/Puerto Rico/8/1934 (H1N1) (PR8) and theA/Hong Kong/1968 (H3N2) (HK68) hemagglutinins in place of the respectivenucleotide sequences encoding amino acids 53 to 276. These three linkerpeptides were selected to have a range of flexibilities, with 4Gpredicted to be the most flexible and PG the most rigid. To test whetherinsertion of a linker in the absence of a disulfide bond at thisposition would yield a more stable product, three additional constructsin the context of the PR8 HA were designed: sequences encoding one, twoor three glycines were inserted in place of the sequences encoding aminoacids 52 to 277 (that is, both the cysteines and the connecting loopwere replaced). Based on the hypothesis that glycosylation may improvetrafficking through the Golgi, the insertion of a glycosylation site(NAS) in place of amino acids 52 to 277 in both the PR8 and the HK68backgrounds was also tested. Finally, a series of three constructs weremade in each of the PR8 and the HK68 HAs in which existing wild-typeamino acids were directly linked: amino acid 51 to 278, 51 to 279, or 50to 280. Constructs were made in the context of an H1 (representative ofgroup 1) and an H3 (representative of group 2) HA since the activity ofneutralizing antibodies targeting the stalk region appears to be limitedto HA subtypes within the same major phylogenetic group (Ekiert, D. C.,G. Bhabha, M. A. Elsliger, R. H. Friesen, M. Jongeneelen, M. Throsby, J.Goudsmit, and I. A. Wilson. 2009. Antibody recognition of a highlyconserved influenza virus epitope. Science 324:246-51; Kashyap, A. K.,J. Steel, A. F. Oner, M. A. Dillon, R. E. Swale, K. M. Wall, K. J.Perry, A. Faynboym, M. Ilhan, M. Horowitz, L. Horowitz, P. Palese, R. R.Bhatt, and R. A. Lerner. 2008. Combinatorial antibody libraries fromsurvivors of the Turkish H5N1 avian influenza outbreak reveal virusneutralization strategies. Proc Natl Acad Sci USA 105:5986-91; Okuno,Y., Y. Isegawa, F. Sasao, and S. Ueda. 1993. A common neutralizingepitope conserved between the hemagglutinins of influenza A virus H1 andH2 strains. J Virol 67:2552-8; and Sui, J., W. C. Hwang, S. Perez, G.Wei, D. Aird, L. M. Chen, E. Santelli, B. Stec, G. Cadwell, M. Ali, H.Wan, A. Murakami, A. Yammanuru, T. Han, N. J. Cox, L. A. Bankston, R. O.Donis, R. C. Liddington, and W. A. Marasco. 2009. Structural andfunctional bases for broad-spectrum neutralization of avian and humaninfluenza A viruses. Nat Struct Mol Biol 16:265-73).

These constructs are summarized in the Table 6 below.

TABLE 6 Summary of Constructs HA1 N-terminal HA1 C-terminal NameStem Segment Linker Stem Segment HA2 Domain PR8-2G SEQ ID NO: 34 Gly-GlySEQ ID NO: 50 SEQ ID NO: 66 PR8-4G SEQ ID NO: 34 Gly-Gly-Gly-GlySEQ ID NO: 50 SEQ ID NO: 66 (SEQ ID NO: 319) PR8-PG SEQ ID NO: 34Pro-Gly SEQ ID NO: 50 SEQ ID NO: 66 PR8-No Cys-1G SEQ ID NO: 177 GlySEQ ID NO: 226 SEQ ID NO: 66 PR8-No Cys 2G SEQ ID NO: 177 Gly-GlySEQ ID NO: 226 SEQ ID NO: 66 PR8-No Cys 3G SEQ ID NO: 177 Gly-Gly-GlySEQ ID NO: 226 SEQ ID NO: 66 PR8-No Cys SEQ ID NO: 177 direct bondSEQ ID NO: 226 SEQ ID NO: 66 PR8-No Cys Δ1 SEQ ID NO: 178 direct bondSEQ ID NO: 227 SEQ ID NO: 66 PR8-No Cys Δ3 SEQ ID NO: 179 direct bondSEQ ID NO: 228 SEQ ID NO: 66 PR8-No Cys NAS SEQ ID NO: 177 Asn-Ala-SerSEQ ID NO: 226 SEQ ID NO: 66 PR8-CON-A SEQ ID NO: 309 Gly-Gly-Gly-GlySEQ ID NO: 310 SEQ ID NO: 66 (SEQ ID NO: 319) HK68-2G SEQ ID NO: 36Gly-Gly SEQ ID NO: 52 SEQ ID NO: 68 HK68-4G SEQ ID NO: 36Gly-Gly-Gly-Gly SEQ ID NO: 52 SEQ ID NO: 68 (SEQ ID NO: 319) HK68-PGSEQ ID NO: 36 Pro-Gly SEQ ID NO: 52 SEQ ID NO: 68 HK68-No CysSEQ ID NO: 183 direct bond SEQ ID NO: 232 SEQ ID NO: 68 HK68-No Cys Δ1SEQ ID NO: 184 direct bond SEQ ID NO: 233 SEQ ID NO: 68 HK68-No Cys Δ3SEQ ID NO: 185 direct bond SEQ ID NO: 234 SEQ ID NO: 68 HK68-No Cys NASSEQ ID NO: 183 Asn-Ala-Ser SEQ ID NO: 232 SEQ ID NO: 68 HK68-CON-ASEQ ID NO: 308 Gly-Gly-Gly-Gly SEQ ID NO: 52 SEQ ID NO: 68(SEQ ID NO: 319)

Expression of Headless HA Constructs in Transfected Cell Cultures

As a preliminary test of protein integrity and stability, levels of theheadless HA constructs expressed in transiently transfected cells wereassessed by Western blotting. As shown in FIG. 11A, headless HAconstructs based on the PR8 HA protein were expressed to levelscomparable to the corresponding full length protein at 24 hourspost-transfection. Within the panel of constructs tested, those whichretained cys 52 and 277 and carried the linker peptides 2G, 4G or PGexhibited the highest steady state levels. In the context of the HK68 HA(FIG. 11B) similar results were seen: all HK68 headless HAs tested weredetected using an antibody specific to the stalk domain, and thosecarrying the 2G, 4G or PG linker between cys 52 and cys 277 were themost abundant. For both HK68 and PR8, the least abundant constructs werethose with the direct linkage between amino acids 50 and 280 and thosewith the inserted glycosylation site (FIG. 11).

To test whether the headless HA constructs were also being transportedto the cell surface, FACS analysis of transiently transfected 293T cellswas performed following surface staining with HA2-specific antibodies.Only the 2G, 4G and PG constructs, which showed high levels by Westernblotting, were tested in this assay. As shown in FIG. 12, the three PR8and the three HK68 based constructs were detected, indicating thattransport through the Golgi to the cell surface was not disrupted by theremoval of the globular head domain. No marked differences among thethree linker bridges were noted in either the Western blotting or FACSbased assays. The constructs carrying the 4G linker bridge were selectedfor further characterization.

Incorporation of Headless HA into Viral Like Particles

As a further test of the functionality of the headless HA molecules,their ability to bud from the cell surface to produce virus likeparticles (VLP) was assessed. While transient expression of the headlessHA constructs alone in 293T cells was not found to result in VLPproduction, co-transfection with an HIV Gag-based construct did lead tothe production of headless HA-containing particles. Specifically, wheneither the PR8 or HK68 4G headless HA constructs was co-expressed in293T cells with a Gag-EGFP (enhanced green fluorescent protein) fusionprotein, particles capable of sedimenting through a 30% sucrose cushionand containing headless HA proteins were released into the cell culturemedium (FIG. 13). Similar results were obtained in the presence (as inFIG. 13) or absence of exogenous trypsin. Unlike the full-length HAprotein, and as expected based on the lack of a globular head domain,the release of headless HA containing particles was not found to bedependent on the presence of neuraminidase activity.

Vaccination with the PR8 Headless HA Provides

Protection Against Homologous Challenge in Mice

The potential of vaccination with a headless HA construct to induce aprotective immune response was evaluated in the mouse model. Athree-dose vaccine regimen was followed in which mice received plasmidDNA on days 0 and 21 and VLP preparations delivered with Freund'sadjuvant on day 56. Each DNA vaccine comprised pGagEGFP alone or incombination with a protein expression vector encoding the full lengthPR8 HA, the PR8 4G headless HA or the HK68 4G headless HA and wasadministered intramuscularly with electroporation. For a final boost,VLP preparations with an HA content of 150 ng (or an equivalent amountof Gag-only VLP) were combined with Freund's complete adjuvant andadministered intraperitoneally to each mouse. On day 77, mice werechallenged intranasally with PR8 virus and then monitored daily formorbidity and mortality for 10 days. In the Gag-only vaccinated group,three out of four mice lost >25% of their initial body weight and weretherefore scored as dead and the fourth animal was seen to lose 15% bodyweight. By contrast, all mice vaccinated with the PR8 4G headless HAsurvived and experienced a maximum, on average, of only 6% weight loss(FIG. 14).

Vaccination of Mice with the PR8 Headless

HA Elicits Cross-Reactive Anti-Sera

The reactivity of serum collected from vaccinated mice against influenzavirus HA proteins was assessed by hemagglutination inhibition (HAI)assay and ELISA. As expected based on the absence of a globular headdomain in the vaccine constructs, the pooled sera from mice immunizedwith Gag alone, the HK68 4G headless HA or the PR8 4G headless HA didnot show HAI activity against PR8 virus prior to challenge. In contrast,pre-challenge sera obtained from mice that received the full length PR8HA vaccine, as well as all post-challenge sera, were strongly reactiveagainst PR8 virus in the HAI assay (Table 7).

TABLE 7 Lack of Hemagglutination Inhibition Activity in Immune Sera ofHeadless HA Vaccinated Mice. Fold-Increase Over Gag-Only Pre-ChallengeSerum Vaccine Pre-Challenge Post-Challenge Gag-only — 8 HK68 4G headlessHA plus Gag 1 8 PR8 4G headless HA plus Gag 1 8 PR8 full length HA plusGag ≧128 ≧128

By ELISA, pre-challenge sera were tested against a panel of HAsubstrates in order to evaluate the breadth of reactivity (FIG. 15).Against concentrated PR8 virion (FIG. 15A), Gag-only and HK68 4Ganti-sera showed only a low level of background activity at the lowestdilution (1:50), while sera from the PR8 full length HA vaccinatedanimals gave a positive signal at a 1:6250 dilution. Antisera againstthe PR8 4G headless HA were less potent than those against the fulllength HA, but reacted positively at a 1:50 dilution. When testedagainst recombinant HA proteins derived from a recent seasonal H1N1(A/New Caledonia/20/1999; FIG. 12B) and a 2009 pandemic H1N1(A/California/04/2009; FIG. 15C) influenza virus, Gag-only and HK68 4Ganti-sera were negative, while sera from mice that received the fulllength PR8 HA were either negative or showed a low level of reactivity.On these heterologous H1 substrates, the PR8 4G anti-sera showed thehighest level of reactivity, with the sera from two of the five mice inparticular demonstrating high titers. Similar results were seen withrecombinant HA proteins of the H2 and H5 subtypes: against theA/Singapore/1/1957 (H2N2) and A/Viet Nam/1203/2004 (H5N1) HAs, seraderived from PR8 4G vaccinated mice showed moderate to high activity,while sera from the remaining groups (including the full length HA) werelargely negative (FIGS. 15D and 15E). Finally, against the H3 subtype HAof A/Hong Kong/1/1968 (H3N2) only the HK68 4G anti-sera produced apositive signal (FIG. 15F). Thus, overall, sera obtained from micevaccinated with the headless PR8 HA showed greater activity againstheterologous strains than did sera from full length PR8 HA vaccinatedanimals. While serum titers of PR8 4G vaccinated mice appeared to behigher against the heterologous HA proteins than against the homologousPR8 virus, a direct comparison should not be made due to differingsubstrates used (purified HA versus whole virus). Within the PR8 4Ggroup, the sera from two mice in particular consistently showedrelatively high titers by ELISA. These serological findings correlatedwith the protection data in that these same two mice were fullyprotected from disease while their three remaining counterparts eachexhibited some weight loss after challenge.

6.2.3 Conclusion

This example describes influenza hemagglutinin (HA) stem domainpolypeptides (“headless HA constructs”) which lack the highlyimmunogenic globular head of the HA protein and are thereby designed topresent the conserved HA stalk region to immune cells. These headless HAconstructs can be stably expressed in mammalian cells and targeted tothe cell surface in a similar manner to full length HA polypeptides.Immunization of mice with a PR8-based HA stem domain polypeptide inplasmid DNA and VLP formats provided full protection against death andpartial protection from disease following a lethal homologous challenge.

Serological analysis revealed that the PR8 4G influenza headless HAconstruct, but not the full-length PR8 HA vaccine, induced antibodieswhich are cross-reactive among group 1 HA subtypes. This findingsuggests that the globular head domain of an intact HA molecule inhibitsrecognition of the stem region by immune cells, either through stericshielding or due to the immune dominance of the membrane distal portionof the protein. These data furthermore suggest that vaccination with anheadless HA construct can lead to protection against divergent influenzastrains.

6.3 Example 3: Challenge with Heterologous Viruses

The data described in Example 2 above shows that mice vaccinated with aPR8 headless HA construct are protected against challenge with PR8 virus(that is, protected against homologous challenge). These data indicatethat an influenza virus hemagglutinin stem domain polypeptide (sometimesreferred to herein as a “headless HA”) is sufficiently immunogenic toact as a vaccine but do not provide information on the breadth ofprotection achieved. To test whether an influenza virus hemagglutininstem domain polypeptide can elicit an immune response which will protectagainst challenge with a range of heterologous viruses, mice will bevaccinated through intraperitoneal injection of 5 μg of a purifiedinfluenza virus hemagglutinin stem domain polypeptide or, as a control,5 μg of full length HA in the context of whole inactivated influenzavirus preparations. In both cases, the vaccine will be combined withMF-59 adjuvant prior to administration. At three weeks post-vaccination,groups of 8 mice will be challenged by intranasal inoculation with 10MLD50 (50% mouse lethal dose) of the virus strains identified in Table8. Mice will be monitored daily up to 14 days post-challenge for changesin body weight and death. The influenza virus hemagglutinin stem domainpolypeptide vaccines are expected to provide superior protection fromdeath and disease following heterologous virus challenges compared tothe conventional whole inactivated virus vaccines.

TABLE 8 Summary of Challenge Experiments Mouse Vaccine Challenge VirusModel^(a) PR8 4G A/Puerto Rico/8/1934 (H1N1) C57BL/6 headless HAA/Netherlands/602/2009 (novel H1N1) DBA-2 A/Viet Nam/1203/2004(H5N1)^(b) C57BL/6 PR8 virus A/Puerto Rico/8/1934 (H1N1) C57BL/6A/Netherlands/602/2009 (novel H1N1) DBA-2 A/Viet Nam/1203/2004 (H5N1)C57BL/6 HK68 4G X31 (H3N2) DBA-2 headless HA A/Rhea/North Carolina39482/1993 (H7N1) DBA-2 X31^(c) virus X31 (H3N2) DBA-2 A/Rhea/NorthCarolina 39482/1993 (H7N1) DBA-2 ^(a)The strain of inbred mouse to beused is based on the lethality of the challenge viruses. Virus strainsthat are less pathogenic to mice must be used in the more susceptibleDBA-2 model. ^(b)Rather than the wild-type virus, a reassortant viruscarrying the HA and NA genes of A/Viet Nam/1203/04 and the remaining sixgenes from PR8 virus will be used. In addition, the multibasic cleavagesite in the HA segment of this virus is mutated to a low pathogenicform. These changes do not affect the antigenicity of the HA protein.^(c)X31 is a mouse adapted virus carrying the HA and NA genes of A/HongKong/1/1968 (H3N2) virus and the remaining six genes from PR8.

All publications, patents and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference. Although the foregoinginvention has been described in some detail by way of illustration andexample for purposes of clarity of understanding, it will be readilyapparent to those of ordinary skill in the art in light of the teachingsof this invention that certain changes and modifications may be madethereto without departing from the spirit or scope of the appendedclaims.

What is claimed is:
 1. A method of inducing an immune response to aninfluenza virus hemagglutinin (HA) in a subject comprising administeringto a subject a vaccine comprising a polypeptide, wherein saidpolypeptide comprises: (a) an influenza hemagglutinin HA1 domain thatcomprises an HA1 N-terminal stem segment covalently linked to a linkerof 1 to 50 heterologous residues that is in turn covalently linked to anHA1 C-terminal stem segment; said HA1 domain in tertiary or quaternaryassociation with (b) an influenza hemagglutinin HA2 domain, wherein thevaccine induces antibodies which are cross-reactive among HA subtypes,and wherein said polypeptide comprises a disulfide bridge between A_(p)and A_(q), wherein A_(p) is Cys that corresponds to amino acid position52 of an HA1 domain using H3 numbering and A_(q) is Cys that correspondsto amino acid position 277 of an HA1 domain using H3 numbering.
 2. Themethod of claim 1, wherein said polypeptide is soluble.
 3. The method ofclaim 2, wherein said polypeptide (a) comprises a thrombin cleavage siteand a foldon domain, and (b) lacks an influenza virus HA signal peptide,transmembrane domain, and cytoplasmic tail.
 4. The method of claim 1,wherein in said polypeptide the HA1 domains contact the HA2 domain. 5.The method of claim 1, wherein in said polypeptide the HA1 C-terminalstem segment is covalently linked to the HA2 domain.
 6. The method ofclaim 1, wherein the polypeptide in said vaccine has a tertiary orquaternary structure having 0-5 Å RMS deviation from the tertiary orquaternary structure of the corresponding polypeptide of 1RUZ.
 7. Themethod of claim 1, wherein the polypeptide in said vaccine selectivelybinds neutralizing antiserum capable of binding an influenzahemagglutinin.
 8. The method of claim 1, wherein the polypeptide in saidvaccine lacks an influenza globular head domain.
 9. The method of claim1, wherein in said polypeptide (i) the amino acid sequences of the HA1domains are at least 70%, 75%, 80%, 85%, 90%, 95%, 96% or 98% identicalto the amino acid sequences of the corresponding domains of an HA1 froman H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 orH16 influenza A virus; or (ii) the amino acid sequence of the HA2 domainis at least 70%, 75%, 80%, 85%, 90%, 95%, 96% or 98% identical to theamino acid sequence of an HA2 from an H1, H2, H3, H4, H5, H6, H7, H8,H9, H10, H11, H12, H13, H14, H15 or H16 influenza A virus.
 10. Themethod of claim 1, wherein in said polypeptide the HA1 N-terminal stemsegment comprises the amino acid sequence A₁₇-A₁₈-(Xaa)_(n)-A₃₈ (SEQ IDNO:146), wherein A₁₇ is Y or H; A₁₈ is H, L, or Q; (Xaa)_(n) representsa sequence of 18-20 amino acid residues; and A₃₈ is H, S, Q, T or N. 11.The method of claim 1, wherein in said polypeptide the HA1 C-terminalstem segment comprises the amino acid sequence A₂₉₁-A₂₉₂, wherein A₂₉₁is T, S, N, D, P or K; and A₂₉₂ is L, M, K or R.
 12. The method of claim1, wherein in said polypeptide the HA2 domain comprises the amino acidsequence A₁₈-A₁₉-A₂₀-A₂₁, wherein A₁₈ is V or I; A₁₉ is D, N or A; A₂₀is G, and A₂₁ is W.
 13. The method of claim 1, wherein in saidpolypeptide the HA2 domain comprises the amino acid sequenceA₃₈-A₃₉-A₄₀-A₄₁-A₄₂-A₄₃-A₄₄-A₄₅-A-₄₆-A₄₇-A₄₈-A₄₉-A₅₀-A₅₁-A₅₂-A₅₃-A₅₄-A₅₅-A₅₆(SEQ ID NO:149), wherein A₃₈ is K , Q, R, L or Y; A₃₉ is any amino acidresidue; A₄₀ is any amino acid residue; A₄₁ is T; A₄₂ is Q; A₄₃ is anyamino acid residue; A₄₄ is A; A₄₅ is I; A₄₆ is D; A₄₇ is any amino acidresidue; A₄₈ is I, V or M; A₄₉ is T, Q or N; A₅₀ is any amino acidresidue; A₅₁ is K; A₅₂ is V or L; A₅₃ is N; A₅₄ is any amino acidresidue; A₅₅ is V, I or L; and A₅₆ is V or I.
 14. The method of claim 1,wherein in said polypeptide said linker is of 1 to 40, 1 to 30 residues,1 to 20 residues, 1 to 10 residues, 1 to 5 residues, 1 to 4 residues, 1to 3 residues, 1 to 2 residues or 1 residue.
 15. The method of claim 1,wherein in said polypeptide said linker is GG, PG, GGG, GGGG (SEQ IDNO:319), GGGGG (SEQ ID NO:320), ITPNGSIPNDKPFQNVNKITYGA (SEQ ID NO:165),or NAS.
 16. The method of claim 1, further comprising administering anadjuvant to the subject.
 17. The method of claim 1, wherein in saidpolypeptide (i) the amino acid sequence of the HA1 N-terminal stemsegment consists of amino acid residues A_(N-term) through A_(p),wherein A_(N-term) is the N-terminal amino acid of a mature HA0 proteinlacking a signal peptide; and (ii) the amino acid sequence of the HA1C-terminal stem segment consists of amino acid residues A_(q) throughA_(C-term) of the HA1 domain, wherein A_(C-term) is the C-terminal aminoacid of the HA1 domain.
 18. A method of inducing an immune response toan influenza virus hemagglutinin (HA) in a subject comprisingadministering to a subject a vaccine comprising a nucleic acid encodinga polypeptide, wherein said polypeptide comprises: (a) an influenzahemagglutinin HA1 domain that comprises an HA1 N-terminal stem segmentcovalently linked to a linker of 1 to 50 heterologous residues that isin turn covalently linked to an HA1 C-terminal stem segment; said HA1domain in tertiary or quaternary association with (b) an influenzahemagglutinin HA2 domain, wherein the vaccine induces antibodies whichare cross-reactive among HA subtypes, and wherein said polypeptidecomprises a disulfide bridge between A_(p) and A_(q), wherein A_(p) isCys that corresponds to amino acid position 52 of an HA1 domain using H3numbering and A_(q) is Cys that corresponds to amino acid position 277of an HA1 domain using H3 numbering.
 19. A method of inducing an immuneresponse to an influenza virus hemagglutinin (HA) in a subjectcomprising administering to a subject a vaccine comprising a viruscomprising a polypeptide, wherein said polypeptide comprises: (a) aninfluenza hemagglutinin HA1 domain that comprises an HA1 N-terminal stemsegment covalently linked to a linker of 1 to 50 heterologous residuesthat is in turn covalently linked to an HA1 C-terminal stem segment;said HA1 domain in tertiary or quaternary association with (b) aninfluenza hemagglutinin HA2 domain, wherein the vaccine inducesantibodies which are cross-reactive among HA subtypes, and wherein saidpolypeptide comprises a disulfide bridge between A_(p) and A_(q),wherein A_(p) is Cys that corresponds to amino acid position 52 of anHA1 domain using H3 numbering and A_(q) is Cys that corresponds to aminoacid position 277 of an HA1 domain using H3 numbering.
 20. The method ofclaim 18, wherein said polypeptide is soluble.
 21. The method of claim19, wherein said polypeptide is soluble.
 22. The method of claim 18,wherein in said polypeptide (i) the amino acid sequence of the HAIN-terminal stem segment consists of amino acid residues A_(N-term)through A_(p), wherein A_(N-term) is the N-terminal amino acid of amature HA0 protein lacking a signal peptide; and (ii) the amino acidsequence of the HA1 C-terminal stem segment consists of amino acidresidues A_(q) through A_(C-term) of the HA1 domain, wherein A_(C-term)is the C-terminal amino acid of the HA1 domain.
 23. The method of claim19, wherein in said polypeptide (i) the amino acid sequence of the HA1N-terminal stem segment consists of amino acid residues A_(N-term)through A_(p), wherein A_(N-term) is the N-terminal amino acid of amature HA0 protein lacking a signal peptide; and (ii) the amino acidsequence of the HA1 C-terminal stem segment consists of amino acidresidues A_(q) through A_(C-term) of the HA1 domain, wherein A_(C-term)is the C-terminal amino acid of the HA1 domain.
 24. The method of claim1, wherein up to 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residuesare deleted from either or both termini of the HA1 N-terminal stemsegment and/or wherein up to 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acidresidues are deleted from either or both termini of the HA1 C-terminalstem segment.
 25. The method of claim 18, wherein up to 10, 9, 8, 7, 6,5, 4, 3, 2, or 1 amino acid residues are deleted from either or bothtermini of the HA1 N-terminal stem segment and/or wherein up to 10, 9,8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues are deleted from either orboth termini of the HA1 C-terminal stem segment.
 26. The method of claim19, wherein up to 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residuesare deleted from either or both termini of the HA1 N-terminal stemsegment and/or wherein up to 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acidresidues are deleted from either or both termini of the HA1 C-terminalstem segment.
 27. The method of claim 1, wherein 1, 2, or 3 residues areadded to a C-terminus of the HA1 N-terminal stem segment and/or wherein1, 2, or 3 residues are added to an N-terminus of the HA1 C-terminalstem segment.
 28. The method of claim 18, wherein 1, 2, or 3 residuesare added to a C-terminus of the HA1 N-terminal stem segment and/orwherein 1, 2, or 3 residues are added to an N-terminus of the HA1C-terminal stem segment.
 29. The method of claim 19, wherein 1, 2, or 3residues are added to a C-terminus of the HA1 N-terminal stem segmentand/or wherein 1, 2, or 3 residues are added to an N-terminus of the HA1C-terminal stem segment.